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United States Patent |
5,239,066
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August 24, 1993
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Yersinia ail nucleic acids
Abstract
Nucleic acids encoding all or part of a Yersinia ail gene are provided. The
nucleic acid comprises at least 50 base pairs of a Yersinia ail gene in
isolated form or consists or a fragment consisting essentially of at least
50 base pairs but not more than 50 kilo base pairs of a Yersinia ail gene.
Such nucleic acids can also be operably linked to transcriptional and
translational initiation and termination sequences which are functional in
a microorganism host.
Inventors:
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St. Geme, III: Joseph W. (Redwood City, CA);
Falkow; Stanley (Portola Valley, CA);
Isberg; Ralph (Brookline, MA);
Miller; Virginia (Van Nuys, CA)
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Assignee:
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The Board of Trustees of Leland Stanford Jr. University (Stanford, CA)
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Appl. No.:
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644826 |
Filed:
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January 23, 1991 |
Current U.S. Class: |
536/23.7; 435/6; 435/69.1; 435/252.3; 435/252.33; 435/320.1; 536/24.32 |
Intern'l Class: |
C12N 015/31; C12N 001/21; C12Q 001/68 |
Field of Search: |
536/27,23.1,23.7,24.32
435/172.3,320.1,69.1,69.3,6,252.3
935/9,11
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References Cited
Other References
Miller, V. L. 1992, ASM News, vol. 58, pp. 26-33.
Miller, V. L. et al., 1988, Infection and Immunity, vol. 56, pp. 1242-1248.
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Primary Examiner: Schwartz; Richard A.
Assistant Examiner: Mosher; Mary E.
Attorney, Agent or Firm: Flehr, Hohbach, Test, Albritton & Herbert
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of application Ser. No.
07/559,904 filed Jul. 30, 1990, abandoned, which is a continuation-in-part
of application Ser. No. 07/340,375, filed Apr. 19, 1989, pending, which is
a continuation-in-part of application Ser. No. 06/761,222 filed Jul. 31,
1985, abandoned.
Claims
What is claimed is:
1. An isolated nucleic acid comprising at least 50 base pairs of a Yersinia
ail structural gene.
2. The nucleic acid of claim 1 wherein said ail structural gene is the
Yersinia enterocolitica ail structural gene.
3. A nucleic acid encoding a Yersinia ail structural gene or fragment
thereof, said nucleic acid consisting essentially of at least 50 base
pairs and not more than 50 kilo base pairs.
4. The nucleic acid of claim 3 wherein said ail structural gene is the
Yersinia enterocolitica ail structural gene.
5. An isolated nucleic acid comprising transcriptional and translational
initiation and termination regions functional in a microorganism host
operably linked to a region encoding the expression product of a Yersinia
ail gene.
6. The nucleic acid of claim 5 wherein said ail gene is the Yersinia
enterocolitica ail gene.
7. A nucleic acid comprising transcriptional and translational initiation
and termination regions functional in a microorganism host operably linked
to a region encoding the expression product of a Yersinia ail gene,
wherein said initiation region is other than the wild type region of said
Yersinia ail gene.
8. The nucleic acid of claim 7 wherein said ail gene is the Yersinia
enterocolitic ail gene.
9. An isolated nucleic acid comprising transcriptional and translational
initiation and termination regions functional in a microorganism host
operably linked to a region encoding a fragment of the expression product
of a Yersinia ail gene, wherein said fragment is encoded by at least 50
base pairs.
10. The nucleic acid of claim 9 wherein said ail gene is the Yersinia
enterocolitica ail gene.
11. A nucleic acid comprising transcriptional and translational initiation
and termination regions functional in a microorganism host operably linked
to a region encoding a fragment of the expression product of a Yersinia
ail gene, wherein said fragment is encoded by at least 50 base pairs, and
wherein said initiation region is other than the wild type region of said
Yersinia ail gene.
12. The nucleic acid of claim 11 wherein said ail gene is the Yersinia
enterocolitica ail gene.
13. A nucleic acid comprising a Yersinia ail structural gene, wherein said
nucleic acid is contained within a microorganism other than the
microorganism from which said ail structural gene is derived.
14. The nucleic acid of claim 13 wherein said ail structural gene is
associated with its native transcriptional and translational initiation
and termination signals.
15. The nucleic acid of claim 13 wherein said ail structural gene is the
Yersinia enterocolitica ail structural gene.
Description
INTRODUCTION
Technical Field
The field of this invention concerns compositions related to invasive
capability of microorganisms.
Background
For many species of microorganisms, invasion and survival within mammalian
cells is central to establishing a successful host-parasite relationship.
This localization within host cells may protect the microorganism to cross
epithelial barriers and subsequently become systemically distributed. The
precise mechanisms by which bacteria enter host tissues have been unclear.
The invasive character of pathogens, while deleterious to the health and
viability of host cells, does provide a mechanism for transfer of
molecules and aggregates across an intact cellular membrane. Thus, if the
invasive quality could be transferred to an innocuous microorganism
strain, such a strain could serve as a vehicle for transporting molecules
of interest into the cytoplasm and organelles of host cells.
There is a further consideration in that the microorganism could provide
for the transfer of genetic material into the mammalian host cell. In this
manner, novel genetic capabilities could be imparted to the host cell. One
capability of interest would be the synthesis of surface membrane proteins
or envelope proteins of pathogens. These proteins would then serve as
antigens to provide a strong immune response, without the host having to
suffer the effects of infection by the pathogen.
Relevant Literature
The invasion of epithelial cells by Yersinia pseudotuberculosis is reported
by Bovallius and Nilson, (1975) Can. J. Microbiol. 21:1997-2007 and Bolin
et al., (1982) Infect. Immun. 37:506-512. The factors associated with
Shigellae invasiveness are described by Hale et al., (1983) Infect. Immun.
40:340-350. Sansonetti et al., (1983) ibid 39:1392-1402 and Maurelli et
al., (1985) ibid 49:164-171 describe the manipulation of the plasmid in
Shigellae encoding functions essential for invasiveness.
Isberg and Falkow, (1985) Nature 317:262-264 and Isberg et al., (1987) Cell
50:769-778 describe the invasion locus of Yersinia pseudotuberculosis
Falkow et al., reviews of infectious diseases, 9 Supp. 5 S450-S455 (1987)
describes the Yersinia gene inv. Miller and Falkow, (1988) Inf. and Imm.
56:1242-1248 describe a second invasion gene named ail (for attachment
invasion locus). Miller et al., Science 243:916-922 describe factors
involved with virulence of bacterial pathogens. Finlay et al., Science
243:940-943 describe invasion gene of Salmonella.
The nucleotide sequence of the Y. enterocolitica ail gene is described in
Mill et al., (1990) Bacteriology 172:1062-1069. See also Miller et al.,
(1989) Infect. and Immun. 57:121-131. For a review of bacterial invasive
strategies, see Finlay and Falkow (1989) 53:210-230.
SUMMARY OF THE INVENTION
Methods and compositions are provided for introducing macromolecules into
mammalian host cells using substantially non-pathogenic microorganisms to
which have been imparted novel invasive capability. The genetic capability
of invasiveness is transferred from one microorganism host to another,
whereby the recipient may now invade mammalian cells and be used as a
carrier for various molecules In this manner, molecules which cannot
normally cross the cell membrane may be transported across the membrane
while retaining an intact membrane and a viable host cell. In one
application, the modified unicellular microorganism may be used as a
vaccine by providing genetic capability for expressing surface membrane
proteins or envelope proteins of various pathogens. A protocol is provided
for screening invasive pathogens for the genetic sequences associated with
such capabilities and transferring such genetic capabilities to
non-pathogenic (innocuous) hosts.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The subject invention provides means for introducing exogenous molecules
into mammalian cell hosts employing unicellular microorganisms as the
vehicle. The method involves isolating the genetic capability of
invasiveness from an invasive unicellular microorganism and transferring
the genetic capability to a recipient unicellular microorganism which does
not naturally have the transferred gene imparting invasive capability,
making any additional modifications in the modified recipient
microorganism, as appropriate, and contacting mammalian cells susceptible
to invasion by the modified recipient microorganism with the modified
unicellular microorganism. A screening protocol is provided for
identifying the genetic region associated with invasiveness, which region
may be introduced directly into the recipient microorganism or may be
further modified prior to introduction. The recipient microorganism may be
used as a vehicle for introducing various molecules, particularly
macromolecules, into a mammalian host susceptible to invasion.
The first aspect of the subject invention to be considered will be the
selection protocol for identifying the genetic capability of invasiveness.
An invasive unicellular microorganism is selected, the genome fragmented,
either mechanically or by partial or complete digestion with one or more
restriction enzymes, and the fragments joined to an appropriate
replication system for introduction into a non-invasive unicellular
microorganism. The replication system may be derived from a plasmid or a
virus, desirably providing for a vector having a copy number equal to or
greater than one and the copy number may be 200 or greater.
The vector should neither be lethal to the host nor result in an undue
reduction in viability. Desirably, the vector should provide a marker
which allows for selection of recipient unicellular microorganisms which
include the vector. Various markers are available, such as biocide
resistance, e.g., antibiotic resistance and heavy metal resistance, genes
imparting prototropy to an auxotrophic host, immunity, and the like.
The genomic fragments inserted into the vector will usually be at least
about 2kb and not more than 50 kb, generally ranging from about 5 to 20kb.
Conveniently, a viral vector may be employed which provides for selection
of fragments in the desired range based on the packaging requirements.
While it is not essential to select for the microorganisms which have
received the vector, it is preferable to select for recipient unicellular
microorganisms. Desirably, the vectors should be capable of stable
episomal maintenance or integration into the genome. Where integration is
involved, amplification of the gene is desirable.
The genome of invasive unicellular microorganisms are mechanically sheared
or digested with one or more restriction enzymes, either partially or
completely, to provide fragments in the range of about 2 to 20 kbp. The
fragments are then inserted into an appropriate vector, such as a viral
vector, e.g., cosmid, or plasmid vector, e.g., pBR322. Non-invasive
unicellular microorganisms are transfected or transformed with the vectors
and modified organisms are selected by means of amarker, e.g., antibiotic
resistance. The desired clones are then enriched by the following
procedure.
The surviving organisms are cloned, suspended in an appropriate nutrient
medium and introduced onto confluent layers of invasive susceptible
mammalian cells. The cells are allowed to incubate for a sufficient time,
usually at least about 1 hour, and less than about 12 hours, conveniently
from about 2 to 6 hours, under conditions which maintain viability of the
cells. The monolayer is then stringently but carefully washed under
conditions which remove nonadherent recipient microorganisms, so that only
adherent microorganisms remain bound to the mammalian cell monolayer. The
internalized cells are then released from the monolayer by treatment with
a mild detergent, e.g., a nonionic detergent, generally at a concentration
in the range of about 0.1 to 2%, more conveniently about 0.5 to 1.5%, in
an aqueous medium. Any mild technique which allows for the viability of
the microorganism with release of the microorganism cells from the
mammalian cells may be employed. The released microorganism cells are then
expanded and cloned.
Transposon mapping may be employed for identifying the transposon
insertions which destroy invasive capability. In this manner, the
structural gene with its associated regulatory signals can be mapped to a
particular site on the fragment. Other techniques involve employing
partial digestions, cloning and screening for invasive capability,
followed by sequencing and identifying specific sequences associated with
transcription and translation as indicative of the structural gene and its
associated regulatory signals.
If desired, the regulatory signals, particularly the transcription
initiation signal, may be modified by the addition or substitution of the
native transcriptional initiation region with a transcriptional region
associated with a different gene. In this way, one can provide for low or
high levels of constitutive or inducible expression of the DNA sequence
encoding for invasive capability. Various transcriptional initiation
regions or promoters are available, which are temperature sensitive, are
inducible in the presence of various metabolites or nutrients, and the
like. Therefore, a transcriptional initiation region may be employed which
is regulated by the unicellular microorganism host and the invasive
capability may be activated or inactivated by physically or chemically
changing the environment of the microorganism host. Thus, nutrients and
metabolites such as glucose, tryptophan, histidine, galactose, lactose,
may be employed to induce or repress the expression of the invasive gene
(inv). The inducible transcriptional regulatory region may be selected in
accordance with the mammalian host, depending upon whether the coinducer
or corepressor is naturally found in the mammalian host or can be
administered to the host.
Constructs may then be prepared which may be used for introducing invasive
capability into an appropriate unicellular microorganism host. Depending
upon the purposes for invasiveness, a wide variety of bacterial or
eukaryotic microorganism hosts may be employed. The subject method
provides for introduction of DNA capability into a mammalian cell where
the unicellular microorganism is employed as the vehicle for introduction
of the DNA capability into a mammalian cell. For example, a shuttle vector
may be provided in the invasive microorganism host which has the
capability for replication in the mammalian cell as well as the
unicellular microorganism, where the shuttle vector may exist as an
episomal element or become integrated into the mammalian cell genome. In
this manner, unicellular hosts for cloning may be used directly for the
transfer of DNA into a mammalian cell host with high efficiency. Thus, a
wide variety of genetic capabilities can be introduced into mammalian
hosts, for example, the expression of lymphokines, hormones, enzymes,
surface membrane proteins, and the like, such as interferons,
interleukins, growth factors, hydrolases, oxidoreductases, receptors,
antibodies, histocompatiability antigens, etc.
A second manner in which the invasive organism may be used is as a vaccine.
For this purpose, in addition to the invasive genetic capability, genes
encoding for surface membrane proteins, capsid proteins, or envelope
proteins, singly or in combination, may be introduced into the invasive
modified microorganism host for injection into a mammal to induce an
immune response. The genes encoding for the antigens may be obtained from
a wide variety of pathogens, including viruses, prokaryotes, e.g.,
bacteria, eukaryotes, e.g., fungi, protists, or the like, or such
intermediate species as chlamydia. In many cases, the gene of interest is
known and available or may readily be isolated. Where not known, the
techniques employed for identifying specific structural genes can be
employed for identifying the genes coding for the desired antigen.
A third manner in which the invasive organism may be used is as a vehicle
for the introduction of molecules, particularly macromolecules, into a
mammalian cellular host, either in vitro or in vivo. For example,
cytotoxic resistance provided by an enzyme could be transferred into cells
or a cytotoxic agent, e.g., aminoglycosides, hybritoxins, etc.,
non-cytotoxic to the microorganism, could be introduced into mammalian
cells. Dyes or other contrast agents could be introduced into the cells
for visualization of cell features. Labelled antibodies could be
introduced into the cells to define the location of particular antigens.
Invasion proteins may be used to introduce particles, such as colloidal
particles, liposomes, slowly degrading or slow release particles, cells,
or the like, where the particles may include drugs, dyes, nucleic acid,
antibodies, or other substances which may have physiological activity. The
invasion proteins may be bound non-diffusibly to the particles, either
covalently or non-covalently. The literature has numerous examples of
commercially available cross-linking agents for joining proteins to other
proteins, sugars, synthetic organic polymers, both addition and
condensation, and the like.
Invasion proteins may also be used to bind mammalian cells to a surface.
Thus in cell cultures, cells may be reversibly bound to a surface,
isolated or otherwise be manipulated. Other uses will also be apparent.
A large number of mammalian replication systems and vectors are available,
particularly viral replication systems, such as SV-40, adenovirus, bovine
papilloma virus, etc. See, for example, Southern and Berg, J. Mol. Appl.
Genet. (1982) 1:327-341; and Mulligan and Berg, Proc. Natl. Acad. Sci. USA
(1981) 78:2072-2076, which are incorporated herein by reference.
One technique would involve employing antisera from a mammalian host who
had suffered, particularly was undergoing infection, by the pathogen of
interest. The pathogen in culture could be lysed and immunoprecipitated
with the antisera from the mammalian host and electrophoresed. A cDNA or
genomic library could be prepared from the pathogen. By at least partially
sequencing the immunoprecipitated proteins, amino acid sequences could be
identified, which could be translated into probes. The probes could then
be used for screening the library and identifying sequences complementary
to the probes. Once sequences which hybridize to the probes have been
identified, where the regulatory sequences are recognized by a prokaryotic
host, the prokaryotic host may be transformed with the sequence and the
expression product identified. Where the expression of the structural gene
is regulated by sequences which are not recognized by prokaryotic hosts,
then some manipulation will be required in identifying the sequence coding
for the particular antigen and inserting the sequence into an appropriate
expression vector. A large number of expression vections exist and various
techniques are available for tailoring the structural gene to remove
superfluous DNA sequences, particularly 5' to the structural gene.
Techniques such as resection with Ba131, primer repair, and in vitro
mutagenesis to introduce a convenient restriction site, have all been used
successfully.
Antigens of interest may come from a wide variety of sources, such as
bacteria, such as Bordetella, Salmonella, Neisseria, Pneumococcus,
Shigellae, Yersinia, Cholera, Meningococcus, Listeria, Mycobacterium,
etc.; viruses, such as HTLV-I, -II, and - III, FeLV, HSV-1 and -2,
Adenovirus, Varicella, Vaccinia, Hepatitis, Influenza, Measles, Rubella,
Smallpox, Typhoid, Yellow Fever, etc. fungi, such as Candida, Microsporum,
Tricpohyton, Arthroderma, Cryptococcus, Blastomyces, Histoplasma,
Coccidroides, Paracoccidroides, Aspergillus, Phycomycetes, Sporotorax,
Epidermophyton, etc. other pathogenic microorganisms, such as Chlamydia,
Giardia, etc.
The organisms may be administered in any convenient form as a vaccine.
Normally, physiologically acceptable carriers will be employed, such as
deionized water, phosphate buffered saline (PBS), aluminum hydroxide,
sugar or the like. Usually, the dosage will be determined empirically;
about 10.sup.4 to 10.sup.10 cells will be administered to a human host,
with proportionate administration based on size to other mammalian hosts.
Generally, there will be a first administration, followed by one or more
administrations at two to six week intervals. The particular amount
administered will depend upon a number of factors, such as the viability
of the invasive microorganism in the host, the concentration of the
antigen on the surface of the pathogen, the number of different antigens
which are present, the level of immune response to the particular
antigen(s), and the like. Administration may be orally, by injection,
intravenously, intraarterially, subcutaneously, intraperitoneally, etc.
The manner of administering live vaccines is well established and may be
found in such texts as Basic and Clinical Immunology, eds. Stites, Stobo.
Fudenberg and Wells. 4th ed. Lange Medical Publications, Los Altos,
Calif., 1982.
The gene coding for the invasive genetic capability may come from any
convenient source. A significant number of organisms are known to be
capable of invasion, such as Yersinia, Chlamydia, Legionella pneumophila,
Listeria monocytogenes, Mycobacterium tuberculosis, Haemophilus
influenzae, Mycobacterium leprae, Salmonella typhosa, Salmonella
typhimurium, enteropathogenic E. coli (EPEC strains), Brucella abortus,
Cryptococcus neoformans, Histoplasma capsulation, Candida albicans,
Tripanosoma cruze, Toxaplasma gondi, Leishmania donovani, etc. Thus, the
organisms may be bacterial, fungal or protozoan. Preferred organisms will
be those which provide a single gene which results in invasive capability.
The affinity for the mammalian host receptor should be at least about 0.1.
Of particular interest are Yersinia, Salmonella, and Haemophilus, which
are demonstrated in the Experimental section as paradigmatic of invasive
organisms.
Genes derived from Yersinia species, particularly Y. pseudotuberculosis and
Y. enterocolitica provide a gene referred to as inv with a phenotype INV,
while the latter provides the additional gene ail with a phenotype AIL.
Salmonella typhimurium, and group A meningococci, are found to have
analogs to the inv and/or ail genes. The inv gene is found not to be
homologous to ail. While both genes provide for invasiveness, the host
range of ail is different from the inv genes. These ail genes are found to
be chromosomal, rather than present on plasmids.
The non-invasive host which is modified to become invasive may be
prokaryotic or eukaryotic and will be selected depending upon its ultimate
purpose. Where the organism is to be the vehicle for transfer of DNA into
mammalian cells in culture, then any convenient organism may be employed,
particularly one which may be used for cloning of the DNA to be
transferred. Therefore, many of the strains of E. coli, e.g., K12, may be
employed as the recipient microorganism which is modified to become
invasive. Where the modified microorganism host is to be employed as a
vaccine, the host will normally be selected so as to be innocuous
(nonpathogenic), to be capable of being viable extracellularly for an
extended period of time, preferably at least about 3 days in the
vaccinated host, and to be subject to ready clearance in the vaccinated
host. Desirably, the modified recipient microorganism will be free of
pyrogens, toxins, or other disease symptom causing factors. By innocuous
is intended that regardless of the dose or route, no disease will be
observed with an immunocompetent host. While pathogenic microorganisms may
be employed, particularly attenuated pathogenic microorganisms, these are
not preferred, since there is a possibility of reversion to pathogenicity.
Microorganism hosts which may be used for modification to invasive
capability include besides E. coli, members of the genus Staphylococci,
Pneumococci Streptococci, e.g., mutants, Neisseria, e.g., catarrhalis,
Veillonella, Lactobacilli, Corynebacteria, Clostridia,, Hemophilic
bacilli, Bacteroides, Actinomycetes, Spirochetes, Mycoplasma, etc.
The manner in which the genetic capability for invasiveness is introduced
into the recipient microorganism may be any of the convenient techniques
including transformation, e.g., calcium precipitated DNA, transfection,
transduction, conjugation, fusion, etc. Applicable techniques may be found
in Maniatis et al., A Laboratory Manual, Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y., 1982. As already indicated, a marker will
normally be present which allows for selection of those microorganisms
which have received the invasive genetic capability. The organisms may
then be grown in an appropriate nutrient medium and used as appropriate.
The invasive microorganisms may be used to prepare antisera for passive
immunization. Thus, .gamma.-globulin could be prepared which has
antibodies to a broad spectrum of pathogens and for strains of a
particular pathogen. The .gamma.-globulin may be isolated and purified
from serum by ammonium sulfate precipitation and fractionation according
to known techniques. Administration to a mammalian host will generally be
in amounts of 50 to 500 mg/kg of host in any physiologically acceptable
carrier. Administration will usually be by injection, e.g., intravenously.
The modified recipient microorganism may also be used in assays for
detecting the presence of antibodies to the antigens foreign to the
modified microorganism or the antigens themselves. They also may find use
in competing with the invasive microorganisms so as to be useful for
therapy.
In addition, the subject microorganisms may be used for expression of
proteins expressed by the inv gene. Particularly, by having high copy
number vectors, the modified foreign microorganisms may be harvested,
lysed and the inv antigen isolated by conventional ways, e.g., affinity
chromatography, electrophoreses, chromatography, etc.
Sequences encoding all or a portion of the invasion genes may be used for
diagnosing pathogenicity or virulence. Two fragments of interest from the
ail locus are 0.9kbp AvaI-ClaI fragment from the plasmid pVM103 and a
1.2kb ClaI-AvaI fragment from the same plasmid or fragments thereof of at
least about 50 bp, preferably at least about 100 bp or extensions thereof
including the entire ail coding region or locus. In conjunction with the
ail probes, a probe referred to as Inv-Ent may be employed, which is a
3.6kb ClaI fragment obtained from pVM101, or 50bp fragment thereof,
preferably at least a 100bp fragment thereof The 3.6kb fragment comprises
most of the inv gene from Y. enterocolitica in addition to adjacent
sequences.
Alternatively, a probe referred to as Inv-PSTB may be employed which is a
2.4kb ClaI-XhoI fragment obtained from pRI203. Isberg et al., Cell
50:769-778 (1987). Of particular interest is the use of ail specific
sequences for detecting pathogenicity In pathogenic strains multiple
copies of sequences having homology to AIL-B are frequently observed. With
the Inv probes, a size pattern is observed when the microorganisms DNA is
cleaved with EcoRV. These strains may be divided into types I, II, I/II,
III, IV and V by the size of fragments observed. This restriction enzyme
cuts once within the Inv-Ent probe, but does not cut within the AIL-B or C
probes. The various types are defined in the Experimental section where,
types I and I/II were found to be pathogenic, while the remaining types
were non-pathogenic, with a strong correlation.
To enhance the invasive phenotype, bacteria are grown in an atmosphere of
about 1% oxygen by maintaining a gaseous source of oxygen at that level.
This may be contrasted with young cultures of laboratory grown cells
(which are typically in the mid-log phase of growth) or overnight cultures
(which are typically in the stationary phase of growth). By growing cells
in 1% oxygen the invasive phenotype is encouraged and the resulting cells
may be more readily screened for the presence of the mRNA encoding for
invasiveness, for use as vaccines or for studying the mechanism of the
regulation of the invasive phenotype.
The following examples are offered by way of illustration and not by way of
limitation.
EXAMPLES
Enrichment for Invasive Phenotype
A cosmid bank was constructed (Hohn, Methods in Enzymology (1979)
68:229-309; Koomey et al., Proc. Natl. Acad. Sci. USA (1982) 79:7881-7885)
by ligating a size-fractionated Sau3AI digest of chromosomal DNA isolated
from Y. pseudotuberculosis strain YPIII (p-) (Bolin and Wolf-Watz, Infect.
Immun. (1984) 43:72-78) into the BamHI site of pREG153 (available from Dr.
R. Gill) a cosmid clonng vehicle that encodes resistance to ampicillin and
is further characterized by its low copy number (5-8). Aliquots of the
ligation mixture were packaged in vitro (Enquist and Sternberg, Methods in
Enzymology (1979) 68:281-298) into phage lambda heads with an extract
prepared from MMSA345 (available from Dr. F. Stahl) and then infected into
E. coli K12 strain HB101 (Maniatis et al., A Laboratory Manual), selecting
for ampicillin-resistant transductants. Pooled bacteria were grown
overnight in culture, washed twice in sterile PBS and resuspended to a
density of 5.times.10.sup.8 bacteria ml.sup.-1. The washed bacteria (2 ml)
were then introduced onto a confluent monolayer containing
1.times.10.sup.7 HEp-2 cells grown in RPMI1640 medium (Irvine
Biologicals), and incubated for 3 hours at 36.degree. C. in a 5% CO.sub.2
atmosphere. Monolayers were then washed 10 times with sterile PBS to
remove non-adherent bacteria. Bacterial clones remaining associated with
the monolayer were released with 1% Triton X-100 in deionized water, and
plated for individual colonies on bacteriological medium.
Screen for Invasive Cultures
Saturated cultures of the transformed bacteria were grown at 28.degree. C.,
washed twice in PBS and resuspended to a concentration of 3.times.10.sup.8
bacteria ml-1 Aliquots (50 .mu.l ) of each strain were added to monolayer
cultures of HEp-2 cells seeded at a concentration of 2.times.10.sup.5
animal cells per microtiter well (24 well, Falcon 3047 microtiter dishes)
in RPMI1640 medium. Bacteria (were centrifuged onto the monolayer at
600.times.g (Devenish and Schiemann, Infect. Immun. (1981) 32:48-55), and
the infected cultures were incubated at 36.degree. C. for 3 hours in 5%
CO.sub.2 atmosphere, to allow binding and invasion of bacteria.
Non-adherent bacteria were removed from the monolayer by washing three
times with sterile PBS, and RPMI medium containing 40 .mu.g ml.sup.-1
gentamicin (Sigma Chemical) was added to each microtiter well. The
incubation was continued at 36.degree. C. for 2 hours in the presence of
the antibiotic, before washing the monolayers twice more with PBS.
Internalized bacteria were then released from the monolayers by the
addition of 1% Triton X100 and titered on L agar plates (Miller,
Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., (1972)). The following table indicates the results.
TABLE 1
______________________________________
Enrichment Procedure Yields E. Coli Strains
That Invade Cell Culture Monolayers
Strain % Invasion.sup.d
______________________________________
YPIII(p.sup.-).sup.a
9.0
HB101.sup.b 0.005
HB101 (pINVA2).sup.c
8.3
HB101 (pINVA7) 7.9
HB101 (pINVG10) 8.7
HB101 (pRI203) 9.2
______________________________________
.sup.a Yersinia pseudotuberculosis strain (Bolin et al. supra).
.sup.b E. coli K12 strain HB101.
.sup.c HB101 harboring cosmids that are denoted in parenthesis).
.sup.d Percentage of bacteria added to HEp2 monolayers that resist
treatment by gentamicin.
It was found that 12 of the 22 candidate strains that survived the
enrichment were invasive, based on the above. It is noteworthy that the
efficiency of escape from gentamicin treatment, which may be equated with
bacterial invasion, was similar to that found for the Y.
pseudotuberculosis strain used as the DNA donor.
To determine if isolated bacterial derivatives could invade cultured animal
cells, ultrathin sections of monolayer cells exposed to several of the
bacterial strains were analyzed by electron microscopy (Horwitz, J. Exp.
Med. (1983) 158:1319-1331). Tissue culture cells (Falcon 3046, 6 well
dishes) seeded with 8.times.10.sup.5 HEp-2 cells in RPMI 1640 were
incubated in the presence of 6.times.10.sup.-7 bacteria for 3 hours at
36.degree. C. Monolayers were then washed 10 times with PBS and incubated
for 10 minutes at 37.degree. C. in the presence of PBS containing 0.11 mM
EDTA. The monolayers were gently washed once more with PBS in the presence
of EDTA, suspended in 1 ml of PBS, and pelleted at 600 xg for 10 minutes.
The cell pellets were successively fixed with 2% glutaraldehyde and 2%
osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4), before staining with
uranyl acetate. Samples dehydrated in ethanol were embedded in Spurrs
(Polysciences), thin sectioned, stained successively with 1% uranyl
acetate and lead acetate (Reynolds, J. Cell. Biol. (1963) 17:208-213), and
visualized with a Phillips 201c electron microscope An E. coli K12 HB101
strain is unable to enter HEp-2 cells. In contrast, the same bacterial
strain harboring an intact inv locus showed a large number of bacteria
associated with the animal cell. These bacteria appeared to be both bound
to the outside of the cell as well as present within large endocytic
vesicles. Furthermore, when E. coli HB101 cells were employed which harbor
the pRI203 plasmid with an intact inv locus, invasion was observed,
whereas the same bacteria which harbored the plasmid pRI203.14::Tn5, which
has a Tn5 insertion mutation in the inv locus were compared, no invasion
was observed. Therefore, invasiveness is only observed with a functionally
intact inv locus. The number of intracellular bacteria had substantially
diminished and the invaded cells remained viable.
The locus of the inv gene was established as follows. One plasmid, pINVA2
was analyzed by causing a series of Tn5 insertion mutations in the cosmid
DNA, taking the precaution that each mutation analyzed was the result of
an independent event. Mutations were induced by the kanamycin-resistance
transposon Tn5 via transposition from .lambda.b221rex::Tn5 cI8570am23Pam80
onto pINVA2 according to the method of deBruijn and Lupski, Gene (1984)
27:313-149. To select for insertions onto the cosmid, kanamycin-resistant
colonies were pooled together and infected lytically with .lambda.lac5
imm21cts. The resulting lysate was used to transduce HB101 to simultaneous
kanamycin- and ampicillin-resistance. Such transductants contained
insertions of Tn5 at random sites on the cosmid. Several hundred such
transductants were assayed for invasiveness as described previously, and
the map position of 20 mutants that eliminated invasiveness and 20
insertions that had no effect on the invasive phenotype were determined.
In order to ensure that each insertion was independent, the physical
location of no more than one mutation from each pool was analyzed. The
sensitivity to insertion inactivation of the invasive phenotype was mapped
to a contiguous 3.2kb region, which region was shown to code for a single
large protein which was indicated as being 108kdal by electrophoresis
using myosin, .beta.-galactosidase and phospholipase G as standards.
Demonstration of Two Genetic Loci for Invasion of Epithelial Cells in Y.
Enterocolitica
Materials and Methods
Bacterial Strains and Tissue Culture Cells
Y. enterocolitica 8081c (Portnoy et al., Infect Immun. (1981) 31:795-782)
and E. coli HB101 were maintained at -70.degree. C in Luria broth (LB)
medium containing 25% (vol/vol) glycerol or on LB agar plates. Antibiotics
were used at the following concentrations: ampicillin, 50.mu.g/ml;
chloramphenicol, 50 .mu.g/ml; kanamycin, 40 .mu.g/ml. Human laryngeal
epithelium (HEp-2), Madin-Darby canine kidney (MDCK), and Chinese hamster
ovary (CHO) cells were maintained and prepared for the invasion assay as
previously described (Finlay and Falkow, UCLA Symp. Mol. and Cell. Biol.
(1987) 64:227-243). Human endometrial (HEC-lB) tissue culture cells were
maintained and prepared for invasion assays in the same manner as HEp-2
cells, with the exception that 10% fetal calf serum was used in the HEC-lB
tissue culture medium. Y. enterocolitica 8081c and E. coli HB101 carrying
recombinant plasmids with Y. enterocolitica invasion genes were grown at
28.degree. C. with aeration for 12 to 18 hours in LB. HB101 carrying the
type I pilus clone PSH2 was grown in LB containing chloramphenicol at
37.degree. C. without aeration (Orindorff and Falkow, J. Bacteriol. (1984)
159:736-744). HB101 carrying the PAP pilus clone pPAP5 (Hindberg et al.,
EMBO J. (1984) 3:1167-1173) was grown on tryptic soy agar plates
containing ampicillin at 37.degree. C. HB101 carrying the X-adhesin AFA-1
clone pIL14 (Labigne-Roussel, Infect Immun. (1984) 46:251-259) was grown
on LB agar plates containing ampicillin at 37.degree. C.
Nucleic Acid Preparation and Analysis
High-molecular weight chromosomal DNA was isolated as previously described
(Hull et al., Infect Immun. (1981) 33:933-938). Plasmid DNA was purified
by the alkaline lysis method (Maniatis et al., (1982) Molecular Cloning: A
Laboratory Manual, CSHL, Cold Spring Harbor, N.Y.). DNA restriction
enzymes and bacteriophage T4 DNA ligase were purchased from Bethesda
Research Laboratories, Inc. Calf alkaline phosphatase was purchased from
Pharmacia, Inc. Restriction enzymes, ligase, and phosphatase were used
according to the instructions of the manufactures.
Invasion Assay
Bacteria (2.times.10.sup.7) were added to each well of a 24-well
microdilution dish which had been seeded with tissue culture cells the
previous day as previously described (Finley and Falkow, (1987) supra).
The microdilution plates were centrifuged for 10 minutes at ambient
temperature at 162.times.g and then incubated in a 5% CO.sub.2 incubator
at 37.degree. C. After 3 hours, the tissue culture medium was removed and
the cells were washed three times with phosphate-buffered saline to remove
nonadherent bacteria. Fresh tissue culture medium 100 .mu.g of gentamicin
per ml was then added, and the plates were reincubated as described above.
After 90 minutes, the medium was removed and the cells were washed twice
with phosphate-buffered saline to remove the gentamicin. The tissue
culture cells were then lysed to release intracellular bacteria by adding
0.2 ml of 1% Triton X-100 to each well. After 5 minutes, 0.8 ml of LB was
added; the final concentration of Triton X-100 was 0.2%. The suspension
was then diluted and plated on the appropriate bacteriological medium to
determine viable counts. Viable counts of the initial bacterial culture
were also determined. Results are expressed as follows: %
invasion=100.times.(the number of bacteria resistant to gentamicin/the
number of bacteria added).
The total number of cell-associated bacteria was determined in the same way
as was the number of intracellular bacteria, with the following exception:
after incubation of the bacteria with the monolayer for 3 hours, the
monolayer was washed five times with phosphate-buffered saline. The
monolayer was then disrupted with 1% Triton X-100 as described above, and
viable counts were determined.
Construction of a Chromosomal DNA Library of Y. enterocolitica
High-molecular-weight chromosomal DNA from Y. enterocolitica 8081c was
partially digested with the restriction enzyme Sau3A as previously
described (Maniatis et al., (1982) supra). Fragments 7 to 10 kilobase
pairs in size were isolated by sucrose gradient fractionation and ligated
into pBR322 that had been digested with BamHI and treated with calf
alkaline phosphatase. The resulting ligated DNA was used to transform
competent E. coli HB101 cells. E. coli HB101 transformants carrying
recombinant plasmids were selected on LB agar plates containing
ampicillin.
Stained Samples and Electron Microscopy
Samples of invasion assays were prepared and stained for electron
microscopy as previously described (Isberg and Falkow, Nature (1985)
317:262-264).
Tn5 Mutagenes
Strains bearing insertions of transposon Tn5 into recombinant plasmid
pVM102 were isolated as previously described (deBruijn and Lupski, Gene
(1984) 27: 131-149) .
RESULTS
Cloning Invasion Genes From Y. enterocolitica
Y. enterocolitica 8081c (serotype 08) was used, which had been isolated
from a patient with septicemia, as our prototype strain (Portnoy et al.
(1981) supra). Strain 8081c lacks the 47-megadalton virulence-associated
plasmid, as well as any other plasmid, yet is still able to invade HEp-2
cells as efficiently as is the plasmid-containing strain. This suggests
that, as in Y. pseudotuberculosis, the invasion determinants are
chromosomally encoded. Consequently, to clone the genes from Y.
enterocolitica 8081c which were involved in invasion, a library of
chromosomal DNA from strain 8081c, was first constructed in the plasmid
vector pBR322. This library was used to transform the normally nonadherent
and noninvasive E. coli HB101. Adhesive and invasive clones were selected
by pooling the resulting transformants and infecting a monolayer of HEp-2
tissue culture cells as described above in Materials and Methods. After 3
hours of incubation, the monolayer was washed 15 times with
phosphate-buffered saline and then lysed with 1% Trition X-100 to release
any intracellular or adherent bacteria The suspension was then spread on
LB plates containing ampicillin. Gentamicin was not used during this
enrichment procedure because we felt that invasion may be a two step
process involving at least two loci, one for attachment and a second for
invasion. If this were the case, the attachment factor would be a
prerequisite for identifying a recombinant strain carrying the invasion
gene.
Clones from the enriched population were then tested individually in the
invasion assay. Approximately 50% of the clones that survived the
enrichment were able to invade HEp-2 tissue culture cells to some degree.
E. coli HB101 transformants expressing an invasion phenotype fell into two
classes (Table 2). The first class, represented by clones 6, 8, and 9,
demonstrated a high level of invasion similar to that of wild-type Y.
enterocolitica 8081c and 800- to 1,400-fold higher than the background
level seen with E. coli HB101. The second class, represented by clones 3
and 7, demonstrated a relatively low level of invasion, which was
nevertheless 40- to 100-fold higher than that of HB101.
TABLE 2
______________________________________
Relative Invasion of Y. enterocolitica
Clones Into HEp-2 Cells
Infecting Strain % Invasion
______________________________________
Y. enterocolitica 8081c
26.8 .+-. 3.6
E. coli HB101 0.0075 .+-. 0.0035
HB101 (pBR322-8081c) (clone 3)
0.82 .+-. 0.24
(= pVM103)
HB101 (pBR322-8081c) (clone 7)
0.37 .+-. 0.19
(= pVM102)
HB101 (pBR322-8081c) (clone 6)
6.2 .+-. 2.1
(= pVM101)
HB101 (pBR322-8081c) (clone 8)
10.8 .+-. 0.1
HB101 (pBR322-8081c) (clone 9)
6.9 .+-. 0.32
______________________________________
.sup.a Strains were used to infect a monolayer of HEp2 tissue culture
cells as described in Materials and Methods. Values are the averages of
duplicate samples, with the ranges indicated, and reflect similar results
from several experiments.
Characterization of Y. enterocolitica Invasion Clones
Isolation and characterization of plasmid DNA from clones 6, 8, and 9
indicated that they all contain the same fragment of Y. enterocolitica
DNA. The recombinant plasmid from clone 6 was called pVM101. Southern
hybridization analysis indicated that plasmid pVM101 shares homology with
the inv gene from Y. pseudotuberculosis and thus is a clone of the Y.
enterocolitica inv locus. The homology shared with Y. pseudotuberculosis
inv is not observed at the restriction map level.
Isolation and characterization of plasmid DNA from clones 3 and 7 indicated
that they have overlapping inserts of Y. enterocolitica DNA. These two
plasmids were renamed pVM103 and pVM102, respectively. Plasmids pVM102 and
pVM103, while they share homology with each other, are not homologous to
pVM101, as demonstrated by both Southern hybridization and restriction map
analysis and thus probably represent a new locus involved in invasion.
A high proportion of E. coli HB101 recombinant cells carrying either pVM102
or pVM103 adhered to the HEp-2 cell monolayers. This raised the
possibility that the low level of invasion observed with the quantitative
invasion assay reflected a small proportion of adherent bacteria that were
protected from the gentamicin treatment. To determine whether bacteria
were actually being internalized, electron microscopy was performed on
samples of HEp-2 cells infected with HB101(pVM102). Bacteria were
frequently found in very close association with eucaryotic cell membranes.
A small number of intracellular bacteria were also observed suggesting
that pVM102 confers a phenotype of adhesion and low-level invasion of
HEp-2 tissue culture cells on E. coli HB101. Electron-dense eucaryotic
structures resembling coated pits were frequently found in association
with attached bacteria, regardless of whether the bacteria were Y.
enterocolitica 8081c or E. coli HB101 carrying either of the recombinant
plasmids, pVM101 or pVM102. Whether or not these structures play a role in
microbial attachment or entry or both is not known at this time. Strain
8081c, unlike HB101 carrying either pVM101 or pVM102, is seen almost
exclusively intracellularly after 3 hours of infection. This observation
would suggest either that there are other gene products directly involved
in invasion or that the gene products encoded by pVM101 and pVM102 do not
function as effectively in an E. coli background. Electron microscopy of
negatively stained cultures of HB101 pVM102 and pVM101 do not indicate the
presence of any pilus-like structures on the surface of these bacterial
cells.
The ability of E. coli HB101 carrying several cloned virulence-associated
adhesions to invade HEp-2 cells was tested (Table 3) to determine whether
adherence alone promoted a low level of invasion HB101 harboring
recombinant plasmids encoding type 1 pili (Orndorff and Falkow, (1984)
supra), PAP pili (Lindberg et al., (1984) supra), or X-adhesin AFA-1
(Lahyne-Roussel et al., (1984) supra) was cultured in such a way as to
maximize expression of the adhesin. While all three of these adhesions
promoted adherence of E. coli HB101 to HEp-2 cells, as determined by
examination of Giemsa-stained cover slips and by quantitative assay, the
presence of type 1 pili, PAP pili, or X-adhesin did not confer an invasive
phenotype of E. coli HB101 (Table 3), nor were coated pits seen associated
with the adherent bacteria. This result indicates that the invasive
phenotype of HB101(pVM102) is a specific property of this recombinant
strain and not a manifestation of simple adhesion.
TABLE 3
______________________________________
Effect of Adhesion on Invasion.sup.a
E. coli Strain % Invasion Adhesion Type
______________________________________
HB101 (pBR322) 0.015 .+-. 0
--.sup.b
HB101 (pVM102) 0.770 .+-. 0.03
--
HB101 (pSH2) 0.067 .+-. 0.001
Type 1 pilus
HB101 (pPAPS) 0.020 .+-. 0
PAP pilus
HB101 (pIL14) 0.007 .+-. 0.001
X-adhesion
______________________________________
.sup.a Strains were used to infect a monolayer of HEp2 tissue culture
cells as described in Materials and Methods. Values are the averages of
duplicate samples, with the ranges indicated, and reflect similar results
from several experiments.
.sup.b --, Not known.
Localization of the Invasion Locus of pVM102
Insertions of transposon Tn5 insertions were mapped, and their invasive
phenotypes were determined. Six insertions that eliminated invasion were
clustered to the right of the unique ClaI site. Insertion mutants
defective for invasion were found only in this region, suggesting that
there is only one region on this plasmid that is responsible for both
adherence and invasion. There are only approximately 650 base pairs
between the Inv+insertions flanking the Inv-insertions, suggesting that
this region contains a very small gene(s). We have designated this region
ail for attachment-invasion locus.
Invasion of Other Tissue Culture Cell Lines
One possibility for the role of ail gene(s) is that it defines target cell
types other than those defined by inv, thus giving Y. enterocolitica a
different range of target host cells than might result from the presence
of inv alone. To test this hypothesis, invasion by HB101 carrying either
of the cloned Y. enterocolitica invasio loci, inv or ail, was examined in
several cell lines (Table 4). All cell - lines were invaded by Y.
enterocolitica 8081c, although to various degrees Plasmid pVM101 promoted
only a low level of invasion of MDCK cells by HB101. Plasmid pVM101 caused
HB101 to invade all tested cell lines, except the MDCK cells, at a
relatively high level. HB101(pVM102) invaded the tested cell lines to
different degrees. The recombinant strain HB101(pVM102) did not invade
MDCK cells, invaded HEp-2 and HEClB cells at a low level, and invaded CHO
cells at a high level. This suggests that cell lines may vary in their
capacity to phagocytize bound bacteria and that this variability can be
specific to the invasion factor expressed by the bacteria.
TABLE 4
__________________________________________________________________________
Invasion of Tissue Culture Cell Lines by
Y. enterocolitica and Y. enterocolitica Bacteria in Tissue Culture Cell
lines.sup.a
% Invasion Of:
Infecting Strain
HEp-2 MDCK CHO HEC1B
__________________________________________________________________________
8081c 26.8 .+-. 3.6
0.93 .+-. 0.07
20.4 .+-. 5.9
124.3 .+-. 5.0
HB101 (pBR322)
.sup. 0.0075 .+-. 0.0035.sup.b
<0.0001 0.08 .+-. 0.01
0.005 .+-. 0.001
HB101 (pVM101)
6.2 .+-. 2.1
0.015 .+-. 0.0015
15.2 .+-. 1.3
19.1 .+-. 2.4
HB101 (pVM102)
0.37 .+-. 0.19
<0.0001 12.7 .+-. 0.2
5.5 .+-. 1.2
__________________________________________________________________________
.sup.a Strains were used to infect monolayers of the tissue culture cell
lines as described in Materials and Methods. Values are the averages of
duplicate samples, with the ranges indicated, and reflect similar results
from several experiments.
.sup.b From infection of HEp2 with HB101. Results were similar for HB101
and HB101 (pBR322).
The total number of cell-associated bacteria, both attached and
intracellular, was also determined (Table 5). Although HB101 (pVM102)
invaded HEp-2 cells at only a low level, it bound to HEp-2 cells as well
as if not better than it did to CHO cells and to a slightly higher degree
to both these cell lines than did HB101(pVM101) (Table 5). Indeed, there
is no correlation between number of bacteria bound and number of bacteria
internalized, suggesting that the interaction of these bacteria with
tissue culture cells that leads to internalization involves more than just
attachment to the cell surface.
TABLE 5
______________________________________
Percent Cell-Associated Bacteria in Tissue Culture Cell Lines.sup.a
% Invasion Of:
Infecting
Strain HEp-2 MDCK CHO HEC1B
______________________________________
8081c 67.0 .+-. 4.0
15.0 .+-. 1.7
51.0 .+-. 5.0
104.0 .+-. 5
HB101 11.0 .+-. 1.7
0.34 .+-. 0.07
3.6 .+-. 0.71
7.4 .+-.
(pBR322)
HB101 34.0 .+-. 2.0
4.4 .+-. 0.05
26.0 .+-. 3.0
58.0 .+-.
(pVM101)
HB101 43.0 .+-. 0.2
4.2 .+-. 0.2
37.0 .+-. 4.0
10.0 .+-.
(pVM102)
______________________________________
.sup.a Strains were used to infect monolayers of the tissue culture cell
lines as described in Material and Methods. Values are the averages of
duplicate samples, with the ranges indicated, and reflect similar results
from several experiments.
The above results demonstrate that the invasion loci, designated inv,
allows a uniformly high level of invasion in several tissue culture lines
and the inv gene of Y. pseudotuberculosis and Y. enterocolitica are
homologous. The second invasion locus of Y. enterocolitica is ail.
Bacteria containing ail exhibit several invasion phenotypes, depending
upon which cell line is infected. ail promotes a high level of invasion of
CHO cells and a low to moderate level of invasion of other cell lines
(HEp-2 and HEC-lB) but allows no invasion of MDCK cells. The recombinant
plasmid pVM102 strongly promotes adherence of E. coli HB101 to many cell
lines, including those cell lines for which it does not promote efficient
invasion (e.g., HEp-2). Although E. coli (pVM102) adheres as well to HEp-2
cells as to CHO cells, more intracellular bacteria are found in CHO cells.
The phenotypes of attachment and invasion which are associated with the
presence of pVM102 appear to be encoded by fewer than 650bp of DNA.
Screening of Yersinia for Pathogenesis Materials and Methods
Bacterial Strains and Tissue Culture Cells
Bacterial strains were maintained at -70.degree. C. in Luria broth (LB)
medium containing 25% (vol/vol) glycerol, or on LB agar plates. Yersinia
strains obtained from a variety of sources were tested for invasion in the
tissue culture assay (TCI phenotype) and hybridization to the probes
described below without prior knowledge of their serotype or source to
ensure an unbiased evaluation. E. coli strain HB101 (FhsdS20 (r.sub.B
-,m.sub.B -) recA13 ara14 proA2 lacYl galK2 rpsL20 xY15 mtll) (Bachman,
Bacteriol. Rev. (1972) 36:525-557) carrying the recombinant plasmids
pRI203, pVM101, or pVM103 were maintained on LB agar plates containing 50
.mu.g/ml ampicillin (Ap). Human laryngeal epithelial (HEp-2) cells were
maintained and prepared for the invasion assay as previously described
(Finlay and Falkow, (1987) supra). Yersinia strains were grown at
28.degree. C. with aeration for 12 to 18 hours in LB for the invasion
assays.
Nucleic Acid Purification and Probe Preparation
Chromosomal DNA was isolated as described (Mekalanos, Cell (1983)
35:253-263). Plasmid DNA was purified by a cleared lysate method followed
by CsCl equilibrium density centrifugation as described (Maniatis et al.,
(1982) supra). DNA restriction enzymes were purchased from Bethesda
Research Laboratory, and were used according to the instructions of the
manufacturer. DNA probes were prepared as follows. Plasmid DNA was
digested with the appropriate restriction endonucleases, and the fragments
were separated by electrophoresis through a 0.7% agarose gel. The DNA
fragments were purified from the agarose gel slices using Geneclean
(Bio101, La Jolla, Calif.). The purified fragments were then labelled with
32P by nick translation for use as probes as previously described
(Maniatis et al., Proc. Natl. Acad. Sci. USA (1975) 72:1184-1188).
Southern Hybridization Analysis
Chromosomal DNA was digested to completion with EcoRV, and the fragments
were separated by electrophoresis through a 0.7% agarose gel. The
separated DNA fragments were transferred to nitrocellulose (Schleicher &
Schuell, Keene, NH) as described by Southern. Hybridizations were
performed at either medium or low stringency. Medium stringency conditions
were as follows: the filter is prehybridized for 1 hour at 37.degree. C.
in 35% formamide/4.times.SSC/0.1% SDS/lmM EDTA/1.times.Denhardts The
prehybridization solution is then removed and hybridization solution (same
as prehybridization solution) containing 250 .mu.g/ml calf thymus DNA and
the boiled probe is added to the filter. After hybridization for 12 to 18
hours at 37.degree. C. the filter was washed three times with
5.times.SSC/0.1% SDS at 65.degree. C. for 15 minutes, 10 minutes, and 5
minutes. Then the filter was washed in 2.times.SSC at room temperature for
5 minutes, air dried, and exposed to Kodak XAR-5 film. Low stringency
conditions are the same as medium stringency except that 20% formamide is
used in the prehybridization and hybridization solutions. The filters can
be reused if washed in 0.25 M NaOH at room temperature for 6 to 10 hours,
and rinsed briefly in 2.times.SSC. Probe results presented for a given
strain were obtained using a single filter that was reprobed several
times. This allowed direct comparison of fragments that hybridized to the
various probes used.
Colony blots were hybridized as above Individual colony blots were used for
each probe rather than rehybridizing the same filter.
Tissue Culture Invasion Assay (TCI)
Either quantitative or qualitative TCI assays were performed. The
quantitative assay is as follows: bacteria (approximately
2.times.10.sup.7) were added to each well of a 24 well microtiter plate
which had been seeded with tissue culture cells the previous day as
described (Finlay and Falkow, (1987) supra). The microtiter plates were
centrifuged for 10 minutes/162.times.g/ambient temperature, and then
incubated in 5% CO.sub.2 at 37.degree. C. After 90 minutes the tissue
culture medium was removed, and the cells were washed three times with
phosphate buffered saline (PBS) to remove non-adherent bacteria. Fresh
tissue culture medium containing 100 .mu.g/ml gentamicin was then added,
and the plates were reincubated as above. After 90 minutes the medium was
removed, and the monolayers were washed twice with PBS to remove the
gentamicin. The tissue culture cells were then lysed to release
intracellular bacteria with 0.2 ml of 1% Triton X-100. After five minutes,
0.8 ml of LB was added bringing the final concentration of Triton X-100
to 0.2%. The suspension was then diluted and plated on the appropriate
bacteriological medium to determine viable counts. Viable counts of the
initial bacterial culture were also determined. Results are expressed as
follows:
% invasion=100.times.(# bacteria resistant to gentamicin/# bacteria added)
The qualitative assay was performed as described above. After washing twice
with PBS to remove the gentamicin, the tissue culture cells were lysed to
release intracellular bacteria by adding 0.2 ml of 1% Triton X-100 to each
well. After five minutes, 0.8 ml of LB was added. Fifty microliters of
this suspension was then spread on an LB agar plate. Results are scored as
either TCI+ or TCI-. TCI+ strains give almost confluent growth on the LB
plate after the assay, whereas TCI- strains give a few isolated colonies.
RESULTS
TCI Phenotype
One hundred seventy-seven Yersinia strains were examined for their TCI
phenotype (1 Y. pestis, 10 Y. pseudotuberculosis 149 Y. enterocolitica, 4
Y. frederiksenii, 4 Y. kristensenii, 3 Y. intermedia, 2 Y. aldovae, 4 new
species 9). These strains were isolated in a variety of geographical
locales and over a time span of more than ten years; nine of the Y.
enterocolitica strains were isolated from four outbreaks and thus probably
represent only our distinct isolates. Twenty-six different serotypes are
represented. Forty strains were examined quantitatively and qualitatively.
Values for TCI+ strains using the quantitative assay ranged from 0.6% to
7.8%; values for TCI- strains ranged from 0.014% to 0.023%. In all cases
there was at least a 26-fold difference between the TCI+ and TCI- strains.
Thereafter the qualitative TCI assay was used. No strain gave ambiguous
results; even strains that exhibited extensive cytotoxic activity were
still clearly invasive. The results observed indicate that serotypes that
are commonly found in the clinical setting were TCI+, whereas serotypes
commonly isolated from the environment were TCI-. This result is in
agreement with previous studies that have observed a good correlation
between potential pathogenicity, as defined by serotype, and the ability
to invade tissue culture cells (Lee et al., Can. J. Microbiol. (1977)
23:1714-1722; Schieman and Denenish, Infect. Immun. (1982) 35:497-506).
Probes
Four different probes were used in the experiments. The Inv-Ent probe was a
3.6 kb ClaI fragment purified from pVM101. This probe contained most of
the inv gene from Y. enterocolitica in addition to adjacent sequences; it
is known that DNA to the left of the EcoRV site is not required for inv
activity The Inv-Pstb probe was a 2.4 kb ClaI-XhoI fragment purified from
pRI203 (Isberg et al., Cell (1987) 50:769-778). This fragment contained
only sequences internal to the Y. pseudotuberculosis inv gene. Although
Southern blot analysis indicated Y. enterocolitica and Y.
pseudotuberculosis inv genes were homologous, no similarity was observed
in their restriction maps. Both these inv genes promote a high level of
invasion by E. coli HB101 of a variety of tissue culture cell lines.
Two probes were used from the Y. enterocolitica ail locus, AIL-B and AIL-C.
AIL-B was a 900 bp AvaI-ClaI fragment from pVM103. AIL-C was a 1.2 kb
ClaI-AvaI fragment from pVM103. Genetic analysis with the transposon Tn5
had indicated that DNA contained within the AIL-C probe was required for
the Ail phenotype. No Tn5 insertions eliminating the Ail phenotype have
been isolated in AIL-B. Preliminary DNA sequence data suggests that only
six base pairs of the ail coding sequence are located within AIL-B; the
remainder of the ail coding sequence is contained within AIL-C.
Most blots were also probed with labelled pBR322 (the cloning vector).
Although some strains clearly had homology to pBR322, the hybridizing
fragments were different from those hybridizing to the inv or ail probes.
Location of inv and ail
The inv and ail genes were cloned from Y. enterocolitica strain 8081c.
Strain 8081c lacks detectable plasmid DNA and plasmid associated
phenotypes, but it is possible that the plasmid has integrated into the
chromosome. To determine if inv and ail are truly chromosomal genes rather
than plasmid genes Southern analysis was performed of purified virulence
plasmid DNA and chromosomal DNA from strain 8081c. None of the probes
hybridized to the plasmid DNA indicating that the inv and ail genes are
not located on the plasmid. The restriction endonuclease used to digest
the DNA (EcoRV) cuts once within the Inv-Ent probe so two fragments from
chromosomal DNA were found that hybridized to the Inv-Ent probe. The AIL-C
probe hybridized to a single fragment from chromosomal DNA, but the AIL-B
probe hybridized to several chromosomal DNA fragments. The restriction
endonuclease used (EcoRV) does not cut within the AIL-B probe, therefore a
sequence present within AIL-B is repeated within the chromosome. Although
there are indications that the virulence plasmid may contain a repeated
sequence, it appears to be distinct from the repeated sequence identified
by the AIL-B probe. The results also indicate that inv and ail are
probably single copy loci and, because probes derived from these genes
hybridize to different fragments, are not adjacent to each other on the
chromosome.
Hybridization of AIL-C to DNA from Y. enterocolitica Strains
Many non-pathogenic strains of Y. enterocolitica were TCI-. We supposed
they might be missing essential genes required for invasion, or they might
contain the appropriate gene in a nonfunctional form. To investigate if
these possibilities pertained to ail or inv we did either Southern or
colony blot analysis of the 149 Y. enterocolitica strains. The AIL-C probe
did not hybridize to DNA from all strains. If one compares hybridization
to AIL-C and the TCI phenotype one finds that 85 of 86 TCI+strains had
homology to AIL-C, whereas only 1 of 63 TCI- strains had homology to
AIL-C. Thus there was a better than 98% correlation between the AIL-C and
TCI phenotypes. This result supports the hypothesis that the ail locus
encodes a virulence factor(s).
Hybridization to AIL-B to DNA from Y. enterocolitica Strains
The AIL-B probe like the AIL-C probe did not usually hybridize to TCI-
strains. However, the TCI+ strains can be divided into two distinct
groups--those that showed strong hybridization to AIL-B, and those that
showed weak or no hybridization to AIL-B. Strains which hybridized
strongly to AIL-B always had multiple fragments that hybridized; the
number and size of these fragments varied from strain to strain. Strains
which hybridized only weakly to AIL-B always had only one fragment that
hybridized. This fragment was usually identical to the fragment that
hybridized to AIL-C. Preliminary sequencing results indicate that the ail
coding region extends only 6 bp into AIL-B. This suggests that the weak
hybridization seen to AIL-B in these strains could be due to a short
sequence associated with ail, but that these strains lack sequences
present in AIL-B that are repeated in the chromosome of strains that
hybridize strongly to this probe.
TCI+ strains that hybridized strongly to AIL-B include the serotypes
013a,13b; 018; 020; 021; 04; 04,32; 04,33; and 08. TCI+ strains that
hybridize weakly or not at all to AIL-B include the serotypes 01,2,3; 03;
05,27; and 09. Interestingly this represents a clean division between the
pathogenic "American" serotypes (i.e., those isolated only in North
America), and other pathogenic serotypes isolated in Europe, Japan,
Southern Africa, and Canada with regard to AIL-B phenotype. Although both
groups of potentially pathogenic serotypes have the ail gene, the
non-"American" strains appear to lack a sequence located near ail that is
found repeated in the chromosome of "American" strains.
Hybridization of Inv-Ent and Inv-Pstb to DNA from Y. enterocolitica Strains
The Inv-Ent probe hybridized to DNA isolated from all strains, nevertheless
there were differences between pathogenic and non-pathogenic strains. We
observed several distinct hybridization patterns. We have labelled them
type I, II, I/II, III, IV, and V. Type I strains had 9.5 kb and 4.0 kb
fragments, and type II had 9.7 kb and 3.8 kb fragments, that hybridized to
the Inv-Ent probe. There were 36 strains that were type I, of these 34
were TCI+. Type I/II shared a 9.7 kb fragment with Type II and a 4.0 kb
fragment with type I; all six type I/II strains were TCI+. The remaining
four groups, II, III, IV, and V were all TCI-. Type V shared a 9.5 kb
fragment with type I and in addition had a 5.0 kb fragment that hybridizes
to Inv-Ent. The hybridization of Inv-Ent to type III was weak relative to
the hybridization observed with other strains. The type III pattern
appears identical to the hybridization pattern observed for Y. intermedia.
Type IV is a catch-all for strains with unique hybridization patterns.
Overall there was a correlation between strains that are TCI+ and the type
I or I/II hybridization pattern, while strains that were TCI- exhibit type
II, III, IV, or V hybridization patterns.
Unlike the Inv-Ent probe, the Inv-Pstb probe contained only inv coding
sequence. Therefore fragments that hybridized to Inv-Pstb were those with
sequences related to the inv gene itself. The Inv-Pstb probe hybridized to
all Y. enterocolitica strains tested except for the type III strains. In
each case the fragment that hybridizes to Inv-Pstb is identical to one of
the fragments recognized by Inv-Ent. InvPstb hybridized to the 9.5 kb
fragment of types I and V, and to the 9.7 kb fragment of types II and
I/II. This result indicates that the hybridization of Inv-Ent to these
fragments probably represents homology to inv sequences. Hybridization of
Inv-Ent to other fragments is probably due to homology to DNA sequences
adjacent to inv. The results using the Inv-Pstb probe also suggest that
type III strains do not have an inv gene and that the hybridization
observed with the Inv-Ent probe is due to sequences adjacent to inv.
Alternatively, but less likely, the type III strains have diverged more
from the Y. pseudotuberculosis inv gene than have other Y. enterocolitica
strains. As mentioned above the type III hybridization pattern is like
that seen for Y. intermedia strains, so it is possible that these strains
have been incorrectly speciated.
Hybridization Phenotypes of Strains Isolated During Several Outbreaks
As noted above, there is an extremely good correlation between the ability
to invade tissue culture cells and hybridization to the inv and ail
probes. Is a similar correlation seen between Y. enterocolitica strains
that clearly caused clinical disease and hybridization to the probes? To
address this question we examined strains previously characterized from
outbreaks of yersiniosis. The strains examined included those isolated
from patients and those isolated from asymptomatic individuals at the same
time in the same region. The data indicate that all Y. enterocolitica
isolates that are strongly implicated as the cause of disease were TCI+,
whereas strains isolated from asymptomatic individuals were either TCI- or
TCI+. As were other TCI+ strains, these strains were type I with the
Inv-Ent probe. A strain isolated from an asymptomatic family member of a
patient was TCI- and was type II with the Inv-Ent probe.
The strains isolated from patients are AIL-C+ and AIL-B+. The TCI- strains
were AIL-B- and AIL-C- as was previously observed for other TCI- strains.
The pattern of hybridization of AIL-B to DNA from random Y. enterocolitica
isolates varied from strain to strain. However, strains isolated from the
same outbreak exhibited identical hybridization patterns to the AIL-B
probe. This probe may, therefore, serve as a useful marker for identifying
the source, and following a particular epidemic.
Presence of inv and ail Sequences Among the Yersiniae
Three species of Yersinia are generally recognized as pathogenic for
animals: Y. pestis, Y. pseudotuberculosis, Y. enterocolitica. Several
other Yersinia species have been defined, but these are generally
considered to be non-pathogenic. Are the differences observed, using the
inv and ail probes, between pathogenic and non-pathogenic Y.
enterocolitica also true for the genus Yersinia as a whole? DNA isolated
from all species of Yersinia tested exhibited homology to the Inv-Ent
probe, but only the pathogenic species showed strong homology to the
Inv-Pstb probe. The DNA fragments hybridizing to Inv-Pstb and Inv-Ent for
Y.. pestis, Y. pseudotuberculosis, and Y. enterocolitica were the same,
suggesting these fragments encode the inv gene. In contrast, Y. aldovae,
Y. intermedia, Y. frederiksenii, Y. kristensenii, and new species 9 showed
only very weak hybridization to Inv-Pstb, and the fragments hybridizing to
Inv-Pstb were not the same as those hybridizing to Inv-Ent, suggesting the
hybridization to Inv-Ent may be due to sequences present in the probe
adjacent to inv rather than to inv itself.
The AIL-B and AIL-C probes both hybridized to DNA isolated from Y. pestis,
and Y. pseudotuberculosis as well as to pathogenic Y. enterocolitica. The
fragments that hybridized to these two probes differed though, indicating
either that the AIL-B and AIL-C sequences are separated on the chromosome
of these species (unlike Y. enterocolitica), or that an EcoRV site
separates these sequences. The AIL-B probe hybridized strongly to only one
DNA fragment in Y. pestis and Y. pseudotuberculosis strain YPIII, but
after longer exposure several additional weak hybridization signals could
be observed for Y. pestis. DNA isolated from twenty-three different
species in addition to the Yersinia spp. was also examined for
hybridization to these probes using low stringency conditions. No
hybridization to the Inv-Ent probe was observed, suggesting this probe
contains Yersinia specific sequences. No hybridization to the AIL-C probe
was observed either, suggesting this probe contains DNA sequences specific
to pathogenic Yersiniae. Only DNA isolated from a strain of Klebsiella
pneumonia hybridized to AIL-B; four hybridizing fragments were observed
when digested with the restriction endonuclease EcoRV, suggesting the
sequence is repeated in Klebsiella pneumonia as it is in Y.
enterocolitica.
Hybridization of inv and ail to Y. pseudotuberculosis
As noted above, all four probes used in this study hybridized to DNA
isolated from Y. pseudotuberculosis strain YPIII. To determine if this is
generally true we examined nine other Y. pseudotuberculosis strains
isolated from both humans and animals. All of these strains were TCI+ and
hybridized to all probes. Two different hybridization patterns were
observed with the Inv-Pstb probe, but there was no obvious correlation
between serotype or source, and hybridization pattern. In addition to the
fragment that hybridized to AIL-B in all strains, a few strains also show
four other weakly hybridizing fragments; these fragments are the same for
all strains that exhibit the phenotype Although only a few Y.
pseudotuberculosis strains were examined it appears that the species Y.
pseudotuberculosis is more homogenous with regard to the inv and ail loci
than is Y. enterocolitica.
The above results demonstrate that tissue culture invasiveness is a good
indicator of potential pathogenicity. This has been proposed in several
other studies. Lee Contr. Microbiol. Immunol. (1979) 5:228-233; Lee et
al., Can. J. Microbiol. (1977) 23:17141722; Schiman and Devenish, Infect.
Immun. (1982) 35:497-506; and Une, Microbiol. Immunol. (1977) 21:365-377.
Strains strongly implicated as the cause of outbreaks of gastrointestinal
disease are exclusively TCI+, and strains isolated from healthy
individuals at the same time and locale as strains associated with disease
are often TCI-. These results support the hypothesis that tissue
invasiveness is an important aspect of Yersinia pathogenesis and that the
ability to invade tissue culture cells is a good in vitro assay for the
invasive ability of virulent strains.
The following describes the identification of the ail gene analog in S.
typhimurium.
MATERIALS AND METHODS
Strains and Media. All rich media was Luria broth (LB) and all minimal
media was M9 (Davis, et al., (1980) Advanced Bacterial Genetics, Cold
Spring Harbor Lab, Cold Spring Harbor, N.Y. p.21). The construction of S.
typhimurium strain CS119 pagCl::TnphoA. phoN2 zxx::6251 Tn10d-Cam was
previously described (Fields, et al., (1989) Science 243:1059-1062)
American Type Culture Collection (ATTC) S. typhimurium strain 10428 was
the wild type parent of CC119. Other strains derived from ATCC 10428
included CS018 which is isogenic to CS119 except for phoP105::Tn10d CS022,
phoP-24 (Con) (Miller and Mekalanos (1989) J. Bacteriol: Fields, et al.,
(1989) supra), and CS015 phoP102::Tn10d-Cam. Other wild type strains used
for preparation of chromosomal DNA included S. typhimurium LT2 (ATCC
15277), S. typhimurium Q1 and S. drypool (Dr. J. Peterson U. Texas Medical
Branch, Galveston), and Salmonella typhi Ty2 (Dr. Caroline Hardegree,
Food and Drug Administration). AP activity was screened on solid media
using the chromogenic phosphatase substrate 5-bromo-4-chloro-3-indolyl
phosphate, (XP). AP assays were performed as previously described and are
reported in units as defined by Miller ((1972) Experiments in Molecular
Genetics, Cold Spring Harbor Lab, Cold Spring Harbor, N.Y. pp. 352-355.
Protein electrophoresis and Western blot analysis. One dimensional protein
gel electrophoresis was performed by the method of Laemmli and blot
hybridization using antibody to AP was performed as previously described
(Peterson and Mekalanos (1988) Infect. Immun. 56:2822-2829). Whole cell
protein extracts were prepared from saturated cultures grown in LB at
37.degree. C. with aeration, by boiling the cells in SDS-PAGE sample
buffer (Laemmli (1970) Nature 227:680-685). Two dimensional gel
electrophoresis was performed by the method of O'Farrell. Proteins in the
10% polyacrylamide slab gels were visualized by silver staining (Meril et
al., (1984) Meth. in Enzymology 104:441)
DNA analysis and sequencing. Chromosomal DNA was prepared by the method of
Mekalanos ((1983) Cell 35:253-263). DNA, size fractionated in agarose
gels, was transferred to nitrocellulose (for blot hybridization) by the
method of Southern ((1975) J. Mol. Biol. 98:503-517). DNA probes for
Southern hybridization analysis were radiolabeled by the random primer
method (Feinberg and Vogelstein (1984) Anal. Biochem.) Plasmid DNA was
transformed into E. coli and Salmonella by calcium chloride and heat shock
(Machlachlan and Sanderson (1985) J. Bacteriol. 161:442-445), or by
electroporation using a Genepulser apparatus (Biorad) as recommended by
the manufacturer (Domer et al., (1988) Nucl. Acids Res. 16:6127-6145). DNA
sequencing was performed by the dideoxy chain termination method of Sanger
as modified for use with Sequenase (U.S. Biochemical). Oligonucleotides
were synthesized on an Applied Biosystems machine and used as primers for
sequencing reactions. Specific primers unique to the two ends of Tn PhoA,
one of which corresponds to the alkaline phosphatase coding sequence and
the other to the right IS50 sequence were used to sequence the junctions
of the transposon insertion.
RNA purification and analysis. RNA was purified from early log phase
Salmonella cultures by the hot phenol method (Case et al., Gene
72:219-236), and run in agarose-formaldehyde gels for blot hybridization
analysis (Thomas (1980) PNAS USA 77:5201).
RESULTS
Identification of an 18 kDa protein missing in a PaqC mutant of S.
typhimurium. Strain CS119 was analyzed by two dimensional protein
electrophoresis to detect protein species that might be absent as a result
of the TnphoA insertion. Identification of multiple changes in the protein
profile of CS119 relative to the parent strain supports that the
expression of a large operon or a regulatory locus was altered as a result
of the transposon insertion. Only a single change in protein species was
observed when strains, isogenic except for their transposon insertions,
were subjected to this analysis. A single protein of approximately 18
kilodaltons (kDa) and pI of approximately 8.0 is missing in the strain
with the pagC::TnphoA insertion. This 18 kDa species is also missing in
Salmonella strains with mutations in phoP and phoQ. The size and pI of
this protein was similar to that predicted to be disrupted by insertion of
TnphoA into pagC.
Analysis of the PagC-AP fusion protein. Western blot analysis using
antibody to AP was performed to determine the size of the AP fusion
protein produced by strain CS119 and to determine the amount of S.
typhimurium DNA sequence needed to encode the fusion protein. The PagC-AP
fusion protein is similar in size to native alkaline phosphatase at 45
kDa, indicating that either a small coding sequence of Salmonella DNA is
contributing to the gene fusion, or a large unstable fusion is produced.
The lack of production of the 45 kDa PagC-AP fusion protein in strain
CS018, isogenic to strain CS119 except for a phoP::Tn10d insertion,
further demonstrates the dependence on an intact phoP locus for efficient
expression of pagC.
Cloning of the pagC::TnphoA insertion. Chromosomal DNA was prepared from S.
typhimurium strain CS119 and Southern hybridization analysis was performed
using a gene fragment from TnphoA as a probe. This fragment hybridizes
preferentially to one end of TnphoA if the restriction enzyme BamHI is
used in the analysis (Peterson and Mekalanos (1988) supra). Therefore, a
number of double restriction endonuclease digestions including BamHl were
used to determine a rough physical map of the restriction endonuclease
sites in the region of the pagC::TnphoA fusion. This analysis indicated
that digestion with the restriction endonucleases XbaI and EcoRV yielded a
single DNA fragment that included the pagC::Tnpho.A insertion in addition
to several kilobases of flanking DNA. Therefore, chromosomal DNA from
strain CS119 was digested with XbaI and EcoRV and ligated into the
bacterial plasmid vector pUC19 that had been digested with the restriction
endonucleases XbaI and SmaI. This DNA was electroporated into the E. coli
strain DHS-alpha and colonies were plated onto LB agar containing the
antibiotics kanamycin (TnphoA encoded) and ampicillin (pUC19 encoded). A
single ampicillin and kanamycin resistant clone containing a plasmid
designated pSM100 was selected for further study.
A radiolabeled DNA probe from pSM100 was constructed and used in Southern
hybridization analysis of strain CS119 and its wild type parent ATCC 10428
to prove that we had cloned the pagC::TnphoA fusion. The probe contained
sequences immediately adjacent to the transposon at the opposite end of
the alkaline phosphatase gene (HpaI endonuclease generated DNA fragment
that included 186 bases of the right IS50 of the transposon and 1278 bases
of Salmonella DNA corresponding to the region between 161 and 1439). As
expected, the DNA fragment from the strain containing the transposon
insertion, CS119, was approximately 7.7 kb (size of TnphoA) larger in size
than the fragment from the wild type strain that hybridizes to the probe.
This was consistent with the supposition that we have cloned the
pagC::TnphoA fusion. In addition, a derivative of plasmid pSM100, pSM101
(which did not allow expression of the pagC-phoA gene fusion off the lac
promoter), was transformed into phoP- (strain CS015) and phoN- (strain
CS019) Salmonella strains and the cloned AP activity was found to be
dependent on phoP for expression. Therefore we concluded that the cloned
DNA contained the pagC::TnphoA fusion.
Mapping of restriction endonuclease sites- DNA sequencing and determination
of the pagC gene product. Restriction endonuclease analysis of plasmid
pSM100 was performed to obtain a physical map of the paqC locus, and to
determine the direction of transcription. Appropriate DNA subclones were
generated and sequence was obtained from the junctions of the transposon
and Salmonella DNA. The sequence was also obtained for Salmonella DNA
extending from the PstI site immediately upstream of the fusion to the
EcoRI site downstream of the fusion. The correct reading frame of the DNA
sequence was deduced from that required to synthesize an active AP gene
fusion. The deduced amino acid sequence contains a methionine start codon
33 amino acids from the fusion of PagC and AP. This 33 amino acid pagC
contribution to the fusion protein was consistent with that observed in
Western blot analysis and contains a hydrophobic N-terminal region,
identified by the method of Kyle and Doolittle ((1892) J. Mol. Biol.
157:105-132), that is a typical bacterial signal sequence (Von Heinje
(1985) J. Mol. Biol. 184:99-105). Specifically, amino acid 2 is a
positively charged lysine, followed by a hydrophobic domain and amino acid
24 is a negatively charged aspartate residue. A consensus cleavage site
for this leader peptide is predicted to be at an alanine residue at amino
acid 23 (von Heinje (1984) J. Mol. Biol. 173:243-251). The DNA sequence
also revealed a typical ribosomal binding site at 6-12 nucleotides 5' to
the predicted start of translation. This suggested that the open reading
frame was, in fact, translated and further supported the assumption that
this was the deduced amino acid sequence of the PagC protein interrupted
by the TnphoA insertion. The deduced amino acid sequence of the open
reading frame was predicted to encode a 188 amino acid protein with a
predicted pI=8.2. This was consistent with the 18 kDa protein of pI-8.0,
noted to be absent in the 2-D polyacrylamide gel analysis of strain CS119.
Therefore identification of the pagC gene product appears established. As
documented below, the pagC transcript is approximately 900 nucleotides in
length. This is approximately 300 nucleotides greater than that which is
devoted to pagC coding sequence, and would seem to preclude the pagC
transcript encoding another protein greater than 100 amino acids in size.
Therefore, the identified pagC gene product is likely to be the protein
essential to virulence and macrophage survival The pagC gene may be part
of a larger operon, but if that is the case, it appears to be the last
gene of the operon, and the upstream gene would encode a small protein.
The pagC gene is present in other strains of Salmonella. To test whether
other S. typhimurium strains LT2, and Q1, as well as S. typhi and S.
drypool contained the pagC gene, chromosomal DNA was prepared from these
strains and screened by blot hybridization analysis using a pagC gene
probe. All Salmonella strains examined demonstrated similar strong
hybridization to an 8.0 kb EcoRV-KpnI and a 4.0 kb AccI restriction
endonuclease fragment suggesting that pagC is a virulence gene common to
Salmonella species.
Identification of the pagC encoded RNA. We purified and analyzed RNA from
S. typhimurium strains ATCC 10428 (wild type), CS015 phoP 102::Tn10d-Cam,
and C5022 pho-24 (Con) to determine the size of the pagC transcript as
well as its dependence on phoP for transcription. An approximately 900
nucleotide RNA is encoded by pagC and this RNA is clearly highly expressed
in a strain with a constitutive phenotype of pag activation as compared to
wild type and phoP- bacteria. This suggests, as expected by gene fusion
analysis, that pagC is transcriptionally regulated by the phoP gene
product and not expressed in early log phase cultures grown in rich media.
Similarity of PagC to Ail and Lom. A computer analysis of protein
similarity using the National Biomedical Research Foundation/Protein
Identification Resource (George et al., (1986) Nucl. Acids. Res. 14:11-15)
protein sequence base was conducted to identify other proteins that had
similarity to PagC in an attempt to find clues to the molecular function
of this protein. Remarkably, PagC was found to be similar to a
bacteriophage lambda protein, Lom that has been localized to the outer
membrane in minicell analysis (Reeve and Shaw (1979) Mol. Gen. Genetics
172:243) as well as demonstrated to be expressed by lambda lysogens of E.
coli. The deduced amino acid sequence of the cloned ail gene product of Y.
enterocolitica was determined and found to also be similar to Lom.
Therefore, we performed a protein family sequence alignment using a
computer algorithm and searched for other members of the family in the
data base using a method that has been shown to be more diagnostic for
family membership than searching individual sequences (Smith and Smith
(1990) PNAS USA 87:118-122) However, no other similarities were found.1
Regions of similarity are located not only in the leader peptide and
transmembrane domains but throughout the protein.
Invasion phenotype of wild type pagC Salmonella strains. Because of the
strong similarity of PagC to ail, PagC- and PhoP- strains were examined in
tissue culture models of invasion to assess whether the PagC protein
functions as an invasion factor as well as a macrophage survival protein.
TABLE 6
______________________________________
Invasive phenotype of wild type S. typhimurium strain
ATCC 10428 and pagC and phoP mutantsa.sup.a
% Invasion
Bacterial Strain HEp-2 CHO MDCK
______________________________________
ATCC 10428 (wild type)
6.35 6.55 8.55
CS119 (paqC::Tn phoA
4.73 4.90 6.68
CSO15 (phoP-) 1.87 2.34 4.46
CS022 (phoP constitutive)
1.82 3.18 3.75
______________________________________
.sup.a Salmonella typhimurium strains were grown at 28.degree. C. with
aeration for 12 to 18 h in LB for the invasion assay. Bacteria
(2/10.sup.7) were added to each well of a 24well microdilution dish which
had been seeded with the indicated tissue culture cells the previous day.
The invasion assay was performed as previously described (Miller and
Falkow (1988) Infect. Immun. 56:1242-1248) with the exception that the
microtiter plates were not centrifuged prior to the first incubation.
Values are the averages of duplicate samples, and reflect similar results
from several experiments.
We were unable to detect more than a modest defect in these strains in
these assays. Therefore, we conclude that the PagC protein may not
function as an invasion protein, or is not the only pathway by which
Salmonella invades epithelial cells. This is consistent with the fact that
orally administered phoP- Salmonella strains pass efficiently from the gut
to the liver and spleen.
The above results demonstrate that a number of invasive organisms have one
or more common invasive gene(s) which are homologous in a plurality of
species. That the gene, inv, as well as a second gene, ail, may be readily
transferred to a non-invasive microorganism host to provide for invasion
by such microorganism into mammalian cells. Furthermore, DNA from the
genes may be used for an in vitro assay for determining pathogenicity of
Yersinia species, as well as other species. The invasive genes find
further use in identifying strains, evaluating levels of pathogenicity and
relationships between pathogenicity and invasion genes.
The mammalian cells are able to endocytose the entire microbial cell based
on the presence of a particular structure encoded on the surface membrane
of the microorganism. The invasive phenotype can be used for diverse
purposes, such as the introduction of exogenous DNA or other molecules
into a mammalian host, induction of an immune response t one or a
plurality of antigens associated with pathogens, so as to be useful as
vaccines, for production of antiserum having a spectrum of antibodies to a
spectrum of pathogens, and for the production of proteins which may be
used to inhibit invasion of pathogens in mammalian host cells.
Studies on the Effects of Growth Conditions on the Adherence and
Invasiveness of Salmonella
Bacterial Strains and Growth Conditions
Salmonella choleraesuis strain SL2824 (Nnalue, N. A. & Stocker, B. A. D.
(1986) Infect. Immun. 54, 635-640) and Salmonella typhi strain 404Ty were
obtained from B.A.D. Stocker (Stanford University School of Medicine,
Calif.). 404Ty is a .DELTA.aroA148 derivative of an Indonesian strain
3083/30 originally characterized by L. LeMinor (Institut Pasteur, Paris,
France) which has two flagellar antigen phases, d and z66 (Guinee, P. A.
M., Jansen, W. H., Mass, H. M. E., LeMinor, L. & Beaud, R. (1981) Ann.
Microbiol. (Inst. Pasteur) 132A, 331-334). Salmonella typhimuirum strains
TN1909 and TN1910, obtained from Charles Miller (Case Western Reserve
University, Ohio), are isogenic strains containing the oxrA+ and oxrAl
loci, respectively. S. typhimurium strain LT2 (DB21)) was obtained from
David Botstein (Genentech, Calif.). Salmonella were grown in LB broth and
on LB agar media 9GIBCO, Grand Island, N.Y.), alone or supplemented with
2,3-dihydroxybenzoic acid DBH) (10 .mu./ml) (Sigma, St. Louis, Mo.) which
is required for optimal growth of the xaroA mutant strain.
Cultures used in some experiments (Table 7 and Table 8, Expt. III) were
grown in LB broth containing antifoam (Sigma Chemical Co., St. Louis,
Mo.). The presence of antifoam does not affect the induction or assay of
bacterial invasiveness (data not shown). Initially, a culture was grown to
logarithmic phase in LB broth containing antifoam while bubbled with air
from the laboratory air lines for 21/4hours. This culture was diluted to
prepare three cultures which were gently bubbled with different gas
mixtures, 5% CO.sub.2 /95% N.sub.2, 1% O.sub.2 /5% CO.sub.2 or 20% O.sub.2
/5% CO.sub.2 /75% N.sub.2 (Liquid Carbonic Specialty Gas Corp., San
Carlos, Calif.). After 3 hours of additional growth, the final culture
density of each culture was -4.times.10.sup.8 CFU/ml (colony-forming units
per ml) (Table 7, Expt. I) and -2.times.10.sup.8 CFU/ml (Table 7, Expt.
II).
Non-agitated cultures were prepared by inoculation of 2 ml of LB broth in
16.times.150 mm borosilicate tubes with 0.04 ml of a stationary phase
culture. For determining invasiveness, as a percent of inoculum surviving
gentamicin, separate cultures were prepared to allow uninterrupted
incubation of each sample. It was characteristic of such cultures that,
during the initial 3 hour incubation at 37.degree. C., the culture density
increased from 10.sup.8 CFU/ml to 4.times.10.sup.8 CFU/ml Bacterial growth
was much slower during the next 4 hours; the culture density after 7 hours
of growth was only 7.times.10.sup.8 CFU/ml. We considered that bacteria
from such cultures were in the late logarithmic phase of growth.
Agitated cultures were prepared by inoculation of 1 or 2 ml of LB broth in
16.times.150 mm borosilicate tubes with a stationary phase culture (a
1:1,000 or 1:50 dilution factor) and placing the tubes on a roller drum
(New Brunswick Scientific, Edison, N.J., 80rpm) at 37.degree. C. Under
these growth conditions, logarithmically growing cultures had <10.sup.9
CFU/ml For the experiment concerned with a time course of invasiveness,
separate cultures were prepared to allow uninterrupted incubation of each
sample.
Overnight cultures were prepared as a source of bacteria in stationary
phase by inoculation of 2 ml of broth with <10.sup.6 CFU and growth with
agitation, except for that used for the experiment determining entry of
bacteria S. choleraesuis of MDCK cells. In this case, a culture was grown
overnight without agitation from a heavy inoculum, as previously described
(Finlay, B. B. & Falkow, S. (1988) Biochimie 70: 1089-1099). Results were
similar to those obtained when stationary phase bacteria from an agitated
culture were used instead
Strains were grown at 37.degree. C. with the exception of strains TN1909
and TN1910 which carry a temperature-sensitive Mu dl prophage; they were
grown at 30.degree. C. CFU were determined by plating serial dilutions
onto LB agar medium.
MDCK cells and growth conditions.
Strain 1 MDCK cells (Gumbiner, B. & Simons, K. 1986) J. Cell Biol. 102:
457-468.) were grown in MEM Eagle medium with Earle's salts
(Cellgro/Mediatech, Washington, D.C.), 5% fetal bovine serum (GIBCO, Grand
Island, N.Y.), without antibiotics. Monolayers for adherence and invasion
assays were prepared by seeding approximately 10.sup.5 cells into each
well of a 24-multiwell tissue culture plate and incubating overnight. MDCK
cells between passage 40 and 60 were used for all assays except for those
in Table 7 and Table 8 (Expt. III) when cells between passage and 10 were
used. The absolute values for bacterial adherence and invasion were
ten-fold lower when MDCK cells between passage 8 and 10 were used vs. when
those between passage 40 and 60 were used. It is possible that a property
of the MDCK cells changed after many serial subcultures resulting in an
increased efficiency of bacterial adherence and invasion The conclusions
of this study appear to be unaffected by this potential change in MDCK
cells.
Adherence assay
The medium overlying the MDCK monolayers was replaced with 1 ml of ice-cold
tissue culture medium and the multi-well dish was placed in an ice-water
bath for 30 minutes prior to the assay. Bacterial samples were diluted if
necessary in LB broth to approximately 10.sup.8 to 4.times.10.sup.8 CFU/ml
so that addition of 40 .mu.l of each bacterial sample to the MDCK
monolayers represented an initial inoculum of 5.times.10.sup.6 to
2.times.10.sup.7 CFU. Control wells without mammalian cells were similarly
prepared to quantitate non-specific bacterial adherence to plastic. The
bacteria were allowed to adhere on ice for 60 minutes and then each well
was rinsed five times with 1-2 ml of ice-cold phosphate-buffered saline
(PBS). Adherent bacteria were released by incubation with 0.2 ml 0.1%
Triton X-100 (Sigma Chemical Co., St. Louis, Mo.) for 5-10 minutes. 0.8 ml
LB broth was then added and each sample was vigorously mixed using a
pasteur pipet. Adherent bacteria were quantitated by plating for CFU on LB
agar media.
In order to measure internalization of adherent bacteria, bacteria were
allowed to adhere as described above. After removal of non-adherent
bacteria with the PBS washes, 1 ml of pre-warmed tissue culture medium was
added to each assay well and internalization of bacteria was allowed to
proceed for 60 minutes at 37.degree. C. in a 5% CO.sub.2 incubator.
Intracellular bacteria were quantitated after incubation with gentamicin
as described below.
Invasion assay
Prior to all invasion assays, the medium overlying each MDCK monolayer was
replaced with 1 ml of pre-warmed tissue culture medium. Bacterial samples
were then added as described above. Invasion assays were incubated at
37.degree. C. in a %5 CO.sub.2 incubator. Assays were terminated by
replacing the overlying medium with 1 ml of tissue culture medium
supplemented with 100 .mu.g/ml gentamicin. Gentamicin, an aminoglycoside
antibiotic, kills extracellular bacteria while intracellular bacteria
remain viable (Vaudaux, P. & Waldvogel, F. A. (1979) Antimicrob. Agents
Chemother. 16: 743-749.). After incubation with the gentamicin for 120
minutes at 37.degree. C. in a 5% CO.sub.2 incubator, the cell monolayers
were washed once with PBS and the viable intracellular bacteria were
released by incubation with 0.2 ml 1% Triton X-100 for 5-10 minutes.
Samples were vigorously mixed with 0.8 ml LB broth using a pasteur pipet.
Viable bacteria were quantitated by planting for CFU on LB agar media.
For the experiment described above, initially tissue culture medium
inoculated with 1.4.times.10.sup.7 CFU of S. choleraesuis from a
stationary phase culture was placed either on MDCK cell monolayers or in
empty wells. In the first case, the entry of Salmonella into MDCK cells
was measured hourly by replacing the overlying medium with tissue culture
medium supplemented with 100 .mu.g/ml gentamicin, as described above. In
this case, calculations were performed to determine the incremental
increase in the number of bacteria that entered the MDCK cells during each
one hour period. The invasiveness of the bacteria incubated in tissue
culture medium alone was determined by transferring the infected medium
onto MDCK cell monolayers at different times. In this case, the number of
bacteria that were able to enter the cells during a one hour assay was
determined, as described above.
Biosynthetic radiolabelling of Salmonella proteins
Bacterial cultures were grown at 37.degree. C. for 5 hours to a final
culture density of approximately 10.sup.7 CFU/ml Cultures containing
tissue culture media were grown in a 5% CO.sub.2 incubator. Bacteria from
0.4 ml of each culture were washed with 1 ml of PBS and then resuspended
in 80 .mu.l M9 medium (GIBCO Laboratories, Grand Island, N.Y.) containing
0.1% glucose and 15 .mu.Ci [.sup.35 S] methionine (Amersham Corp.,
Arlington Heights, Ill.). Samples were incubated at 37.degree. C. for 1
hour and then centrifuged to harvest the bacteria. The samples were
analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis
(Laemmli, U.K. (1970) Nature (London) 227: 680-685.).
RESULTS
Effect of growth conditions on Salmonella invasiveness
Previous work has suggested that Salmonella is not inherently able to
adhere to or enter mammalian cells (Finlay, B. B., Heffron, F. & Falkow,
S. (1989) Science 43: 940-943.). Salmonella which were grown to
logarithmic phase in LB broth were also found to be inherently
non-adherent (Finlay, B. B., Gumbiner, B. & Falkow, S. (1989) J. Cell
Biol. 107: 221-230, Finlay, B. B., Heffron, F. & Falkow, S. (1989) Science
243,:940-943.). Experiments to measure the invasiveness of Salmonella that
were grown to logarithmic and late logarithmic phase were performed. The
results indicate that the invasiveness of bacteria grown under these
different conditions varied dramatically. In order to understand this
variation, experiments were performed so as to more carefully measure the
invasiveness of bacteria during the growth of a culture. Non-agitated and
agitated LB broth cultures were examined.
Non-agitated cultures of S. choleraesuis were prepared by inoculation of 2
ml of LB broth with 0.04 ml of a stationary phase culture (a 1:50 dilution
factor) at t.sub.o. Bacteria grown overnight to stationary phase were not
able to enter MDCK cells during a one hour assay. However, after growth of
bacteria in non-agitated LB broth for 3 to 7 hours, a 100-fold increase in
bacterial invasiveness was observed.
Agitated cultures of S. choleraesuis were prepared by inoculating 2 ml of
LB broth with a stationary phase culture (a 1:1,000 or 1:50 dilution
factor) at t.sub.o. When the ability of bacteria from agitated culture to
enter MDCK cells was measured, dramatic differences were seen depending on
the stage of growth of the bacteria sample. S. choleraesuis from cultures
in logarithmic phase were not maximally invasive. However, when the
culture was in late logarithmic phase, bacterial invasiveness increased at
least 10-fold. The bacterial culture then lost invasiveness in stationary
phase which is consistent with previous observations that bacteria grown
overnight in LB broth to stationary phase are non-invasive. Similar
results were also found for non-agitated and agitated cultures of S.
typhi.
The dramatic differences in the invasiveness of Salmonella from different
cultures were not affected by inhibition of bacterial growth and de novo
protein synthesis during the assay period. Thus, in the assay performed,
the difference in the ability of Salmonella to enter MDCK cells reflects
an inherent property of the bacterial inoculum.
Effect of oxygen availability of Salmonella invasiveness
The above experiments show that S. choleraesuis and S. typhi from
non-agitated cultures and agitated cultures in late logarithmic phase are
at least 10-fold more invasive than those from agitated cultures in
logarithmic phase.
A possibly significant difference between these two growth conditions that
may account for the differences in Salmonella invasiveness is the
availability of oxygen (Strauch, K. L., Lenk, J. B., Gamble, B. L. &
Miller C. G. (1985) J. Bacteriol. 161: 673-680, Khosla, C. & Bailey, J. E.
(1988) Nature 331: 633-635.).
The amount of oxygen available during bacterial growth was varied by
bubbling gas mixtures containing different amounts of oxygen into the
culture medium. Unlike the non-agitated and agitated cultures, these
bubbled cultures are more easily compared with one another since they can
be grown with similar mixing and to similar culture densities. S.
choleraesuis and S. typhimurium were grown with gases containing 0% or 1%
oxygen were 6- to 70-fold more invasive than those grown with gas
containing 20% oxygen (Table 7). These results confirm that Salmonella
invasiveness is induced by oxygen-limited growth conditions.
TABLE 7
______________________________________
Effect of oxygen availability on S. choleraesuis and S.
typhimurium invasiveness
Bacterial Bacterial Bacterial
Expt. #
species culture invasiveness (%)**
______________________________________
I S. choleraesuis
non-agitated,
100 .+-. 5
late log
agitated, 8 .+-. 0.4
mid-log
0% 0.sub.2 75 .+-. 0
1% 0.sub.2 62 .+-. 4
20% 0.sub.2
13 .+-. 3
I S. typhimurium
non-agitated,
100 .+-. 0
late log
agitated, 0.1 .+-. 0
mid-log
0% 0.sub.2 41 .+-. 1
1% 0.sub.2 218 .+-. 18
20% 0.sub.2
3 .+-. 0.3
______________________________________
*Bacterial cultures were grown as described previously.
**The ability of bacteria to enter into MDCK cells (passage 8 to 10)
during one hour was measured as described previously. Assay values
represent the average of two separate assays and were normalized such tha
the invasiveness of bacteria from the nonagitated cultures equals 100%.
The actual percent of the inoculum from the nonagitated cultures that
entered the MDCK cells was 1.6% and 1.7% for Expts. I and II,
respectively.
Invasiveness of a S. typhimurium mutant defective in anaerobic regulation
33 proteins of S. typhimurium that increase in synthesis in response to
anaerobiosis have been detected by two-dimensional gel electrophoresis and
subsequently identified (Spector, M. P., Aliabadi, Z., Gonzaler, T. &
Foster, J. W. (1986) J. Bacteriol. 168: 420-424.). The oxrA locus of S.
typhimurium is required for the regulation of 14 of these proteins
(Strauch, K. L., Lenk, J. B., Gamble, B. L. & Miller, C. G. (1985) J.
Bacteriol. 161: 673-680, Spector, M. P., Aliabadi, Z., Gonzaler, T. &
Foster, J. W. (1986) J. Bacteriol. 168: 420-424.). The, OxrA protein is
homologous to the Fnr protein of Escherichia coli which functions as an
oxygen-sensitive transcriptional activator during anaerobiosis (Strauch,
K. L., Lenk, J. B., Gamble, B. L. & Miller, C. G. (1985) J. Bacteriol.
161: 673-680, Sprio, S. & Guest, J. R. (1988) Molec. Microbiol. 2,
701-707, Trageser, M. & Unden, G. (1989) Molec. Microbiol. 3: 593-599.).
Since the expression of Salmonella invasiveness is induced in
oxygen-limited growth conditions, the invasiveness of isogenic oxrA.sup.+
/oxrA.sup.- S. typhimurium strains grown in aerobic and oxygen-limited
conditions was examined. The oxrA+ and oxrA- strains were shown not to
differ in their ability to enter MDCK cells. Both strains exhibited a
100-fold increase in invasiveness when grown in oxygen-limited vs. aerobic
conditions. Thus, the oxrA locus does not regulate the expression of
Salmonella invasiveness.
Adherence and entry of Salmonella choleraesuis to MDCK cells
It has been proposed that Salmonella initially bind to the mammalian cell
surface and are subsequently internalized by a host-direct
endocytic/phagocytic process (Finlay, B. B. & Falkow, S. (1988) Biochimie
70: 1089-1099, Moulder, J. W. (1985) Microbiol. Rev. 49: 98-337.). Thus,
alteration of either the binding properties or the efficiency of the
internalization process would affect bacterial invasiveness. S.
choleraesuis grown in different cultures conditions were examined for
changes in their ability to bind to, and subsequently enter MDCK cells. A
similar assay for adherence and internalization has been used to examine
the interaction between the Yersinia pseudotuberculosis invasion protein
and its mammalian receptor (Isberg, R. R. (1989) Molec. Microbiol. 3:
1449-1453, Isberg, R. R., Voorhis, D. L. & Falkow, S. (1987) Cell 50:
769-778.).
Although S. choleraesuis does not bind efficiently to MDCK cells in this
assay, the data in Table 8 revealed that bacteria from oxygen-limited
cultures were able to adhere to MDCK cells 20- to 100-fold better than
bacteria from aerobic cultures. Bacteria from stationary phase also
appeared to adhere to MDCK cells. Measurement of the number of adherent
bacteria that were able to subsequently enter the MDCK cells demonstrated
that a relatively high percentage of bacteria were internalized in all
cases, except for the bacteria from stationary phase. Adherent stationary
phase bacteria were internalized 100-fold less efficiently, a finding
consistent with previous observations that such bacteria are not invasive.
TABLE 8
______________________________________
Adherence and subsequent internalization of
S. choleraesuis into MDCK cells
Bacterial Internalization of
Bacterial adherence adherent bacteria
Expt. #*
culture** at 0.degree. C. (%)***
at 37.degree. C. (%) .sctn.
______________________________________
I non-agitated,
100 .+-. 15 59 .+-. 7
late log
agitated, 26 .+-. 1 35 .+-. 2
late log
agitated, 0.8 .+-. 0.5
54 .+-. 11
mid-log
stationary 22 .+-. 2 0.3 .+-. 0.1
phase
II non-agitated,
100 .+-. 8 40 .+-. 5
late log
agitated, 40 .+-. 0.5
16 .+-. 9
late log
agitated, 6 .+-. 0.4 15 .+-. 6
mid-log
stationary 83 .+-. 16 0.4 .+-. 0.1
phase
III non-agitated,
100 .+-. 16 7 .+-. 0.1
late log
agitated, 83 .+-. 8 7 .+-. 2
late log
agitated, 58 .+-. 7 9 .+-. 0.3
mid-log
stationary 2 .+-. 1 -
phase
______________________________________
*MDCK cells between passage 40 and 60 were used for Expt. I and II wherea
those between passage 8 and 10 were used for Expt. III.
**Bacterial cultures were prepared as described above.
***Assay values represent the average of two separate assays and were
normalized such that the adherence of bacteria from the nonagitated
cultures equals 100%. The actual percent of the inoculum from the
nonagitated cultures that were adherent was 1.6%, 1.2% and 0.12% for
Expts. I, II and III, respectively.
.sctn. Values represent the average of two separate assays.
Values were too low to allow calculation.
Induction of S. choleraesuis invasiveness in the presence and absence of
MDCK cells
Previous experiments have shown that S. choleraesuis grown overnight in LB
broth is not inherently able to enter MDCK cells. However, after further
incubation with the mammalian cells in tissue culture medium, bacteria
adherence and invasiveness was induced. It has been proposed that
induction of invasiveness might occur in response to exposure to the
epithelial cell surface (Finlay, B. B., Heffron, F. & Falkow, S. (1989)
Science 243: 940-943.). The induction of invasiveness seen in the
experiment in Table 8 may have been due to the presence of the MDCK cells,
or to a change in bacterial growth state during the incubation period.
Experiments to distinguish between these two possibilities were performed
by comparing the invasiveness of S. choleraesuis incubated with MDCK cells
vs. S. choleraesuis incubated identically in tissue culture medium but in
the absence of MDCK cells. The results obtained revealed that regardless
of the presence of MDCK cells, bacterial invasiveness was induced 40-fold
after four hours of incubation. Thus in this in vitro system, induction of
Salmonella invasiveness can be accounted for by growth in tissue culture
medium alone.
Analysis of proteins synthesized by invasive Salmonella
In order to identify the major proteins expressed by invasive Salmonella,
S. choleraesuis and S. typhi grown under different culture conditions were
radiolabelled. Invasive bacteria from non-agitated cultures were compared
with bacteria from agitated culture (which are at least 10-fold less
invasive than the bacteria grown in the non-agitated). Proteins with
approximate molecular weights of 65 Kd and 44 Kd were found to be
preferentially produced by invasive Salmonella. By comparison to
previously published protein patterns, it appears that both of these
proteins have been identified as expressed by Salmonella adherent to MDCK
cells (Finlay, B. B., Heffron, F. & Falkow, S. (1989) Science 243:
940-943.).
The following describes the identification of the inv gene analog in H.
influenzae.
MATERIALS AND METHODS
Bacterial strains and culture conditions
H. influenzae strain SU1 is a clinical isolate which was obtained from the
Clinical Microbiology Laboratory at Stanford University Hospital. It is
nonencapsulated and appears to be a nontypable strain on the basis of
multilocus enzyme electrophoresis. Negative staining electron microscopy
demonstrates that this strain is nonpiliated. It was stored frozen at
-80.degree. C. in brain heart infusion broth with 20% glycerol. For all
assays bacteria were streaked from frozen aliquots onto fresh chocolate
agar supplemented with 1% Supplement VX (Difco Laboratories, Detroit,
Mich.) and were grown at 37.degree. C. in a 5% CO.sub.2 incubator
overnight. For growth in broth, bacteria were suspended from an 18 hour
chocolate agar plate in brain heart infusion broth supplemented with hemin
(10 .mu.g/ml) and NAD (3.5 .mu.g/ml).
Escherichia coli K12 was obtained from the Stanford University collection
and is the Stanford wild type prototrophic strain (ATCC 10798). It was
stored frozen at -80.degree. C. in LB broth with 50% glycerol and was
grown either on LB agar plates or in LB broth (GIBCO, Grand Island, N.Y.).
Tissue culture cells
Chang epithelial cells, Wong-Kilbourne derivative, clone 1-5c-4 (human
conjunctiva) were obtained from the American Type Culture Collection (ATCC
CCL20.2). Cells were maintained in Modified Eagles Medium with Earle's
salts and nonessential amino acids (Irvine Scientific) supplemented with
10% heat-inactivated fetal calf serum (GIBCO, Grand Island, N.Y.) and 2.0
mM 1-glutamine. They were subcultivated every two to five days and were
used between passages three and fifty. For adherence and invasion assays,
approximately 1.5.times.10.sup.5 cells suspended in 0.5 ml of tissue
culture medium were seeded into each well of 24-well tissue culture plates
(Becton Dickinson Labware) which were then incubated at 37.degree. C. in
5% CO.sub.2 for 18 to 20 hours. In the case of radiolabeled adherence
assays, cells were seeded onto 12 mm diameter glass coverslips placed on
the bottom of each well of tissue culture plates.
Adherence Assays
(i) Radiolabeled assay
Bacteria were suspended in supplemented brain heart infusion broth and
grown at 37.degree. C. with aeration to late log phase (10.sup.9 CFU/ml)
They were pelleted, washed once with phosphate buffered saline (PBS), pH
7.0, and resuspended in methionine assay medium (Difco Laboratories,
Detroit, Mich.). After incubation at 37.degree. C. with aeration for 15
minutes, 50 .mu.Ci of [.sup.35 S]methionine (Amersham, Arlington Heights,
Ill.) was added to the suspension along with hemin and NAD and incubation
was continued for another 45 minutes. While there was minimal bacterial
growth during the period of radiolabeling, on average the level of
incorporation of radioactivity was 0.01 cpm/bacterium. Radiolabeled
bacteria were washed three times with PBS, resuspended in PBS, and
inoculated (10 .mu.l, 1-2.times.10.sup.7 CFU) onto monolayers. Tissue
culture plates were incubated at 37.degree. C. (5% CO.sub.2) for various
times. After the desired incubation period, the infected monolayers were
rinsed three times with PBS with gentle rocking to remove nonadherent
bacteria. Nonspecific adherence was determined by inoculating radiolabeled
bacteria onto coverslips without epithelial cells, followed by rinsing
with PBS. Coverslips were transferred to vials containing 5 ml of aqueous
counting scintillant (ACS II; Amersham, Arlington Heights, Ill.) and
counts were determined and compared with the inoculum. Expression of
adherence as percent of original radioactivity provides a measure of
bacterial attachment which is independent of later bacterial events, such
as replication. Calculations of the average number of adherent bacteria
per eukaryotic cell accounted for bacterial replication and assumed no
epithelial cell division during the course of an assay.
(ii) Viable count assay
Bacteria were grown to log phase as for the radiolabeled adherence assay.
Approximately 1-2.times.10.sup.7 CFU (10 .mu.l) were inoculated directly
from the broth culture onto monolayers, and tissue culture plates were
incubated at 37.degree. C. in 5% CO.sub.2. Following the appropriate
incubation period, monolayers were rinsed four times with PBS and then
treated with trypsin-EDTA (0.05% trypsin, 0.5 mM EDTA) in PBS to release
them from the plastic support. Well contents were agitated, and dilutions
were plates on supplemented chocolate agar for H. influenzae or LB agar
for E. coli, yielding the number of adherent bacteria per monolayer.
Invasion Assay
To determine the number of bacteria entering the cells of an epithelial
monolayer we adapted the assay of Isberg and Falkow (Nature (1985)
317:262-64) Briefly, bacterial were grown and monolayers were infected as
outlined above for the viable count adherence assay. After the desired
incubation, monolayers were rinsed three times with PBS, and fresh tissue
culture medium containing gentamicin (100 .mu.g/ml) was added. Tissue
culture plates were incubated for another two hours, rinsed twice with
PBS, then treated with trypsin-EDTA (0.05% trypsin, 0.05 mM EDTA) in PBS.
Well contents were agitated and dilutions were plated to quantitate the
number of internalized bacteria per monolayer.
Outer Member Protein Analysis
Outer membrane fractions were prepared from samples of radiolabeled
bacteria using the microprocedure of Carlone et al. with minor
modifications. (J. Clin. Microbiol. (1986) 24:330-32. Bacteria were
initially pelleted by centrifugation a 5000.times.g and 4.degree. C. for
10 minutes. Pellets were washed once with 1 ml of cold 10mM HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer, pH 7.4, then
resuspended in 1 ml of the same buffer in preparation for sonication.
Samples were sonicated on ice with eight 10 second bursts at maximal
output using a microsonicator and a 2.3 millimeter probe (Kontes). Intact
cells were removed by centrifugation at 15,600.times.g and 4.degree. C.
for 2 minutes in a microcentrifuge (Brinkmann). Supernatants were decanted
into fresh tubes and centrifuged at 15,600.times.g and 4.degree. C. for 30
minutes to pellet bacterial membranes. Pellets were resuspended in 0.2 ml
10 mM HEPES buffer (pH 7.4). Following the addition of 0.2 ml of 2%
sarcosyl (N-lauroyl sarcosine) in 10 mM HEPES buffer (pH 7.4) to
solubilize cytoplasmic membranes, samples were incubated at room
temperature for 30 minutes with intermittent mixing. Insoluble fractions
containing outer membranes were collected by centrifugation at
15,600.times.g and 4.degree. C. for 30 minutes. The outer membranes were
resuspended in equal quantities of distilled water and Laemmli buffer
(Laemmli, Nature (1970) 227:680-685) and were stored at -20.degree. C.
Samples were heated at 100.degree. C. for 5 minutes, and 100,000 cpm were
loaded onto sodium dodecyl sulfate-polyacrylamide gels. Electrophoresis
was performed at a constant current of 25 mA with stacking and resolving
gels which contained 5% and 10% polyacrylamide, respectively. Gels were
stained with Coomassie blue for 30 minutes and were destained overnight.
Autoradiograms were made using XAR-5 film.
Microscopy
(i) Transmission electron microscopy
Chang epithelial cells were seeded into 35 mm.times.10 mm Contur Permanox
tissue culture dishes (Miles Scientific, Naperville, Ill.) and bacterial
were inoculated onto monolayers as above. Following the appropriate
incubation period, monolayers were rinsed four times with PBS and fixed
with 2% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4 at
4.degree. C. for 2 hours. After washing with 0.1 M sodium phosphate
buffer, samples were postfixed with 1% OsO4 in sodium phosphate buffer for
90 minutes and then incubated in buffer overnight. Samples were next
washed with distilled water and stained with 0.25% aqueous uranyl acetate
for 1 hour. They were dehydrated with a series of graded ethanol solutions
and embedded in a firm Spurr resin. Samples were sectioned, stained with
uranyl acetate and lead citrate, and examined in a Phillips 201c electron
microscope.
(ii) Scanning electron microscopy
Monolayers were prepared on glass coverslips as for radiolabeled adherence
assays. They were inoculated with bacteria and incubated for variable
periods of time, followed by rinsing four times with PBS. Samples were
fixed with 2% glutaraldehyde in 0.1M sodium phosphate buffer, pH 7.4 for
two hours, then dehydrated in a critical point apparatus (Polaron). After
a gold evaporation step, samples were examined with a JEOL scanning
electron microscope.
(iii) Light microscopy
Monolayers were prepared on glass coverslips as above. After inoculation of
bacteria and incubation for the desired period of time, monolayers were
rinsed four times with PBS. Samples were fixed with HPLC grade Methanol
for 15 minutes and were then stained with Giemsa stain diluted 1:20 for 30
minutes. They were rinsed four times with distilled water and were mounted
on glass slides for viewing by light microscopy.
RESULTS
Adherence
Using radiolabeled bacteria and measuring adherence as percent of original
radioactivity, we noted minimal adherence by strain SU1 after one hour of
incubation with the epithelial monolayer. However, adherence continued to
increase over time, peaking about ten-fold higher by four to six hour. In
contrast, there was minimal nonspecific adherence with no significant
change over time. E. coli K12 demonstrated negligible adherence which
failed to change appreciably over the course of an assay. Adherence by E.
coli was measured up to four hours; at later time points the monolayer
began to lose integrity. Adherence by H. influenzae strain SUl gradually
increased from 0.5% at one hour to greater than 8% by six hours. Because
of bacterial replication that occurred during the course of the assay, the
actual number of adherent bacteria per eukaryotic cell increased more than
100-fold between hours one and six. The kinetics of adherence appeared
similar for inocular varying between 10.sup.6 and 10.sup.8 CFU.
Examination of Giemsa stained samples and results from the viable count
adherence assay confirmed this gradual increase in adherence over time
with a peak at four to six hours.
This increment in adherence with time suggested a dynamic interaction
between infecting H. influenzae and the epithelial monolayer. To begin to
explore the nature of this interaction, we examined adherence to a
monolayer which had been chemically fixed for two hours with 2%
glutaraldehyde. Fixation virtually abolished bacterial attachment,
implying the importance of viable epithelial cells or a receptor
cross-linked by glutaraldehyde or both. In contrast, treatment of
monolayers with emetine dihydrochloride (Streeter and Rees, J. Cell. Biol.
(1987) 105:507-15), a selective inhibitor of eukaryotic protein synthesis,
had no effect on adherence. In initial studies of bacterial events
occurring during the course of the bacteria-epithelial cell interaction,
we pretreated bacteria with the bactericidal antibiotic gentamicin and
then added these nonviable bacteria to the cell monolayer. Adherence by
nonviable bacteria was markedly reduced compared with viable organisms. By
six hours there were an average of two bacteria per eukaryotic cell.
Incubation at 4.degree. C. produced a similar inhibition of adherence.
Addition of a bacteriostatic concentration of tetracycline (6 .mu.g/ml) to
the monolayers just prior to inoculation of viable bacteria again produced
a marked inhibition of adherence, indicating the need for protein
synthesis at some point following initial exposure to the epithelial cell
monolayer. To extend this observation, we introduced tetracycline
following incubation for one hour and two hours and then measured
adherence at six hours in each case. In the presence of tetracycline there
was minimal further adherence from hour one to six and hour two to six,
respectively, implying a need for ongoing protein synthesis throughout the
period of incubation in order for maximal adherence to occur. Examination
by light microscopy confirmed the results.
The above evidence demonstrating a requirement for ongoing protein
synthesis suggested that an adhesin might be induced during the
incubation. Bacteria sampled at one, two, four, and six hours were unable
to agglutinate human erythrocytes, arguing against pili as the putative
adhesin. Similarly, negative staining electron microscopy on bacteria
examined after incubation for six hours revealed no pili-like structures.
Identification of Candidate Adhesin Molecules
To identify candidate proteins which might be mediating the process of
adherence, we compared profiles of outer membrane proteins synthesized
during incubation with epithelial cells for adherent versus nonadherent
populations of bacteria. In this experiment, methionine free Dulbecco's
MEM was substituted for the usual tissue culture medium and emetine
dihydrochloride was added to monolayers to inhibit selectively eukaryotic
cell protein synthesis. Bacteria were then inoculated onto monolayers and
were radiolabeled with [.sup.35 S]methionine from hours two to four of
incubation. Following the period of radiolabeling, we separated bacteria
remaining in the supernatant (nonadherent) from those associated with the
epithelial monolayer (adherent). As a control we simultaneously
radiolabeled bacteria incubated in tissue culture media alone (i.e., in
the absence of a monolayer). Subsequently we isolated detergent-insoluble
proteins from adherent bacteria, supernatant bacteria, and bacteria
labeled in media alone. To confirm that emetine had completely inhibited
protein synthesis by the epithelial monolayer, we performed the same
procedure on an uninfected monolayer radiolabeled for two hours. This
analysis yielded several notable results. Specifically, adherent bacteria
expressed at least three protein bands which were absent from either
supernatant or control bacteria (molecular weights approximately 16, 20,
and 80 kilodaltons). In addition, there were at least five other protein
bands which were expressed more prominently by adherent bacteria (36, 44,
46, 56, and 72 kilodaltons). Expression of a protein of molecular weight
28 kilodaltons was diminished in adherent organisms compared to
supernatant or control bacteria. Western analysis using an antiserum
directed against a 13-amino acid peptide of H. influenzae type b pilin
(Rabbit antiserum R 13, provided by J. Gilsdorf, Univ. of Michigan School
of Medicine) demonstrated no reactivity with any of these proteins. This
antiserum recognizes pilins from all of the type b and nontypable H.
influenzae strains tested (Gilsdorf et al. Pediatr. Res., (1990) 27:170A).
Invasion.
Bacterial invasion was determined on the basis of the ability to survive
treatment with gentamicin. Gentamicin is a bactericidal antibiotic which
is unable to enter eukaryotic cells. Therefore, bacteria adherent to
epithelial cells are killed by this drug while internalized organisms are
protected from its effects (Kihlstrom and Andaker, J. Antimicro Chemo.
(1985) 15:723-728). Preliminary experiments indicated that a concentration
of gentamicin equal to 100 .mu.g/ml was sufficient to kill 100% of an
inoculum of 5.times.10.sup. bacteria for strain SU1.
Using this assay, strain SUl showed increasing numbers of internalized
bacteria per monolayer over time, rising from 2.times.10.sup.1 at one hour
to nearly 10.sup.5 at five and one half hours. Thus, by this later time
point there was on average approximately one intracellular organism per
epithelial cell in the monolayer. Correcting for bacterial replication by
calculating the ratio of internalized bacteria to total bacteria present
at each time point confirmed an increase with time in the ability of
bacteria to enter. In contrast, invasion by bacteria which were incubated
in the presence of tetracycline was nearly completely inhibited. E. coli
K12, a noninvasive organism, showed virtually no invasion in this assay.
Invasion by E. coli was measured up to four hours; as noted above, at
later time points the monolayer began to lose integrity.
As most invasive bacteria enter eukaryotic cells via a
microfilament-dependent process (Moulder, Microbiol. Rev. (1985)
49:298-337), we examined the effect of cytochalasin D on the process of
invasion by H. influenzae. Cytochalasin D is a potent inhibitor of
microfilament formation which at a concentration of 1 .mu.g/ml prevents
entry of a variety of cell lines by a number of different bacterial
species. Using this concentration of cytochalasin D and an incubation time
of four hours we noted a marked reduction in the number of internalized
bacteria for strain SU1. Interestingly, inhibition of microtubules with
concentrations of colchicine ranging from 0.5 to 5 .mu.g/ml also resulted
in a profound decrease in invasion. There was no effect on adherence by
either cytochalasin D or colchicine.
Microscopy
We used scanning and thin section transmission electron microscopy to
confirm our results with the aforementioned adherence and invasion assays
and to gain additional information about the interaction between infecting
H. influenzae and Chang conjunctival cells. The general sequence of events
was viewed with scanning microscopy. At one hour there were occasional
cells with a few associated bacteria. In each case it was apparent that
initial contact between bacteria and the cell surface occurred via
attachment to microvilli. Most cells were free of adherent bacteria at
this early time point and were morphologically identical to uninfected
monolayers. By two hours a majority of cells had adherent bacteria, and
the number of bacteria per cell was greatly increased compared with one
hour. By four hours there was a further increase in the number of
epithelial cells with associated bacteria and a peak in the number of
bacteria per cell. The six hour samples revealed a slight decrement in
adherence and early evidence of a toxic effect on the epithelium with
beginning loss of microvilli. Higher magnification (20,000 to
30,000.times.) demonstrated no obvious bacterial surface appendages at
either early or late time points.
Transmission electron microscopy also demonstrated that initial contact
with the epithelium involved interaction with microvilli. Subsequently,
bacteria moved into direct contact with the plasma membrane of host cells
and occasionally were seen penetrating this surface. By two to four hours
there were obvious internalized organisms. In general, in a given thin
section invaded cells had one or a few internalized bacteria, and
sometimes bacteria were seen in pairs. It was difficult to resolve
definitively whether internalized bacteria were enclosed by a vacuole,
although occasionally they appeared to be. At six hours there were
numerous cells with one to several internalized bacteria.
All publications and patent applications cited in this specification are
herein incorporated by reference as if each individual publication or
patent application were specifically and individually indicated to be
incorporated by reference.
Although the foregoing invention has been described in some detail by way
of illustration and example for purposes of clarity of understanding, it
will be readily apparent to those of ordinary skill in the art in light of
the teachings of this invention that certain changes and modifications may
be made thereto without departing from the spirit or scope of the appended
claims.
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